Tbx5a and Tbx5b paralogues act in combination to control separate vectors of migration in the fin field of zebrafish.

The T-box containing family member, TBX5, has been shown to play important functional roles in the pectoral appendages of a variety of vertebrate species. While a single TBX5 gene exists in all tetrapods studied to date, the zebrafish genome retains two paralogues, designated as tbx5a and tbx5b, resulting from a whole genome duplication in the teleost lineage. Zebrafish deficient in tbx5a lack pectoral fin buds, whereas zebrafish deficient in tbx5b exhibit misshapen pectoral fins, showing that both paralogues function in fin development. The mesenchymal cells of the limb/fin bud are derived from the Lateral Plate Mesoderm (LPM). Previous fate mapping work in zebrafish has shown that wildtype (wt) fin field cells are initially located adjacent to somites (s)1-4. The wt fin field cells migrate in opposing diagonal directions placing the limb bud between s2-3 and lateral to the main body. To better characterize tbx5 paralogue functions in zebrafish, time-lapse analyses of the migrations of fin bud precursors under conditions of tbx5a knock-down, tbx5b knock-down and double-knock-down were performed. Our data suggest that zebrafish tbx5a and tbx5b have functionally separated migration direction vectors, that when combined recapitulate the migration of the wt fin field. We and others have shown that loss of Tbx5a function abolishes an fgf24 signaling cue resulting in fin field cells failing to converge in an Antero-Posterior (AP) direction and migrating only in a mediolateral (ML) direction. We show here that loss of Tbx5b function affects initial ML directed movements so that fin field cells fail to migrate laterally but continue to converge along the AP axis. Furthermore, fin field cells in the double Tbx5a/Tbx5b knock-down zebrafish do not engage in directed migrations along either the ML or AP axis. Therefore, these two paralogues may be acting to instruct separate vectors of fin field migration in order to direct proper fin bud formation.

Facial typicality and attractiveness reflect an ideal dimension of face structure.

Face perception and recognition are important processes for social interaction and communication among humans, so understanding how faces are mentally represented and processed has major implications. At the same time, faces are just some of the many stimuli that we encounter in our everyday lives. Therefore, more general theories of how we represent objects might also apply to faces. Contemporary research on the mental representation of faces has centered on two competing theoretical frameworks that arose from more general categorization research: prototype-based face representation and exemplar-based face representation. Empirically distinguishing between these frameworks is difficult and neither one has been ruled out. In this paper, we advance this area of research in three ways. First, we introduce two additional frameworks for mental representation of categories, varying abstraction and ideal representation, which have not been applied to face perception and recognition before. Second, we fit formal computational models of all four of these theories to human perceptual judgments of the typicality and attractiveness (a strong correlate of typicality) of 100 young adult Caucasian female faces, with the models expressed within a face space derived from facial similarity judgments via multidimensional scaling. Third, we predict the perceived typicality and attractiveness of the faces using these models and compare the predictive performance of each to the empirical data. We found that of all four models, the ideal representation model provided the best account of perceived typicality and attractiveness for the present set of faces, although all models showed discrepancies from the empirical data. These findings demonstrate the relevance of mental categorization processes for representing faces.

Overlooked Symptoms in Autoimmune Hepatitis Negatively Impact Many Facets of Life.

BACKGROUND: Significant reduction in quality of life among patients with autoimmune hepatitis (AIH) patients has been observed in several studies. While acute symptoms associated with AIH have been well described, little is known about the overall impact of living with AIH on patients' quality of life. The aim of this qualitative descriptive study was to describe the impact of AIH and associated symptoms on quality of life from the perspectives of patients living with AIH. METHODS: Patients from Autoimmune Hepatitis Association support groups were recruited to participate in one of five online focus groups conducted between August and September 2020. After enrollment, patients were asked to complete a brief demographic and disease history questionnaire. A single moderator conducted interviews with each group guided by seven questions focused on the impact of AIH on the participants' quality of life. Each session was recorded, transcribed, and verified. Content analysis was used to summarize the participants' responses. RESULTS: The participants' discussed three overarching topics: (a) symptoms of AIH and medication side effects, (b) the impact the disease and symptoms/side effects on five domains of quality of life (work life, relationships with friends and family, social life, leisure activities, and diet and exercise) and (c) interactions with healthcare providers and recommendations for future research. CONCLUSIONS: Living with AIH can have profound effects on patients' quality of life in several domains. Healthcare providers and the AIH research community should focus on developing further strategies that can improve the quality of life in persons suffering from AIH.

Serum C-terminal telopeptide of Type-I collagen (CTx) concentration and myocardial hyperechogenicity in cats with hypertrophic cardiomyopathy.

Objective: This study sought to determine the serum concentrations of C-terminal telopeptide of Type-I collagen (CTx), a marker of collagen degradation, in a hospital population of cats with hypertrophic cardiomyopathy (HCM). The study also evaluated the prevalence of myocardial hyperechogenicity of the left ventricle (LV) in the same cats. Animals and procedure: Cats brought to a university veterinary cardiology service entered the study when they had an echocardiographic diagnosis of HCM; echocardiographically normal cats served as controls. Serum CTx concentrations were assessed using ELISA. Results: There was no difference in serum CTx concentrations between cats with HCM and controls (HCM: median 0.248 ng/mL, controls: median 0.253 ng/mL; P = 0.4). Significantly more cats with HCM (60%) showed echocardiographic LV myocardial hyperechogenicity compared to normal controls (17%; P = 0.0057), but serum CTx concentrations were not different between these 2 groups. Conclusion and clinical relevance: These results indicate that, as in human patients with HCM and in contrast to earlier feline studies, there was no evidence of enhanced collagen degradation indicated by serum CTx concentrations in cats with HCM compared to normal controls.

Concentration sérique de télopeptide C-terminal du collagène de Type I (CTx) et hyperéchogénicité myocardique chez des chats atteints de cardiomyopathie hypertrophique. Objectif: Le premier objectif de cette étude était d'évaluer le taux sérique d'un marqueur de dégradation de collagène, soit le télopeptide C-terminal du collagène de Type-I (CTx), chez les chats atteints de cardiomyopathie hypertrophique (CMH). Le deuxième objectif était d'évaluer la prévalence de l'hyperéchogénicité du myocarde du ventricule gauche chez ces mêmes chats. Animaux et procédures: Les chats participant à l'étude avaient été présentés pour soins à un service de cardiologie vétérinaire universitaire, et ces chats avaient un diagnostic échocardiographique soit de CMH, soit d'aucune lésion cardiaque (groupe témoin). Le taux sérique de CTx a été évalué de façon immuno-enzymatique par ELISA. Résultats: Les résultats n'ont démontré aucune différence entre le taux sérique de CTx chez les chats atteint de CMH et le taux sérique de CTx chez les chats sans lésion cardiaque (CMH : médiane, 0,248 ng/mL; groupe témoin : médiane, 0,253 ng/mL; P = 0,4). Plus de chats atteints de CMH (60 %) que de chats dans le groupe témoin (17 %) ont démontré une hyperéchogénicité du myocarde du ventricule gauche à l'échocardiographie (P = 0,0057), quoique les taux sériques de CTx n'étaient pas différents entre ces 2 groupes. Conclusion et signification clinique: Ces résultats n'indiquent aucune augmentation de la dégradation de collagène chez les chats atteints de CMH, ce qui s'apparente aux résultats provenant d'études antérieures de la CMH chez l'humain mais non pas à ceux provenant d'études de la CMH féline.(Traduit par les auteurs).

Anterior lateral plate mesoderm gives rise to multiple tissues and requires tbx5a function in left-right asymmetry, migration dynamics, and cell specification of late-addition cardiac cells.

In this study, we elucidate a single cell resolution fate map in the zebrafish in a sub-section of the anterior Lateral Plate Mesoderm (aLPM) at 18 hpf. Our results show that this tissue is not organized into segregated regions but gives rise to intermingled pericardial sac, peritoneum, pharyngeal arch and cardiac precursors. We further report upon asymmetrical contributions of lateral aLPM-derived heart precursors-specifically that twice as many heart precursors arise from the right side versus the left side of the embryo. Cell tracking analyses and large-scale cell labeling of the lateral aLPM corroborate these differences and show that the observed asymmetries are dependent upon Tbx5a expression. Previously, it was shown that cardiac looping was affected in Tbx5a knock-down and knock-out zebrafish (Garrity et al., 2002; Parrie et al., 2013); our present data also implicate tbx5a function in cell specification, establishment and maintenance of cardiac left-right asymmetry.

Age-specific impacts of nicotine and withdrawal on hippocampal neuregulin signalling.

Smoking remains the leading cause of preventable death in the United States, with 87% of smokers starting before the age of 18. Age of initiation is a major predictive factor for smoking frequency and successful smoking cessation. People who initiate smoking during adolescences are 2.33 times more likely to become heavy smokers and half as likely to quit compared with smokers who started during adulthood. Additionally, schizophrenia, a disease state linked to altered neurodevelopment during adolescence, is a major predictive factor for smoking status. Smoking rates among people suffering from schizophrenia are between 60% and 90%. Interestingly, the Neuregulin Signalling Pathway (NSP), which plays an important role in neurodevelopment, is implicated in both schizophrenia and nicotine use disorder. Specifically, SNPS in neuregulin 3 (Nrg3) and Erb-B2 Receptor Tyrosine Kinase 4 (ErbB4) have been associated with smoking cessation outcomes and schizophrenia. Here, we examine the effects of chronic nicotine (18 mg/kg/day) and 24-h withdrawal on NSP gene expression in the hippocampus of adult (20-week-old) and adolescent (4-week-old) mice. We show that withdrawal from chronic nicotine decreased the expression of Erbb4 mRNA in the hippocampus of the adult mice but increased the expression of cytosolic Erbb4 protein in adolescent mice. Nrg3 mRNA and protein expression was not altered by chronic nicotine or withdrawal in the adult or adolescent cohorts, but Nrg3 mRNA and synaptosomal protein expression was lower in the adult withdrawal group when compared with their adolescent counterparts. These results highlight the age-specific effects of nicotine withdrawal on the NSP and may contribute to the lower quit rate and higher cigarette consumption of smokers who initiation during adolescences.

WT1 complete gonadal dysgenesis with membranoproliferative glomerulonephritis: case series and literature review.

BACKGROUND: Intronic WT1 mutations are usually causative of Frasier syndrome with focal segmental glomerulosclerosis as the characteristic nephropathy. Membranoproliferative glomerulonephritis is not commonly associated with disorders of sex development but has been recently identified as a WT1-associated nephropathy, but usually in cases of exonic mutations in either isolated Wilms tumor or Denys-Drash syndrome. METHODS: The clinical and genetic data from 3 individuals are reported. RESULTS: This report describes the kidney manifestations in 3 individuals from 2 unrelated families with Frasier syndrome intronic WT1 mutations, noting that 2 of the 3 individuals have histologically confirmed membranoproliferative glomerulonephritis. CONCLUSIONS: These case reports support expansion of the clinical spectrum of the kidney phenotypes associated with Frasier syndrome providing evidence of an association between WT1 mutation and an immune complex-related membranoproliferative glomerulonephritis. A higher resolution version of the Graphical abstract is available as Supplementary information.

Acquired Systolic Dysfunction and Subsequent Congestive Heart Failure Following Treatment of Hypoadrenocorticism in Two Dogs.

Acquired cardiomyopathies have been described in human patients with hypoadrenocorticism. Several mechanisms have been described to explain the cardiac effects of primary adrenal insufficiency, but, clinically, these manifestations may be underappreciated in dogs. In humans, there is an infrequently described, reversible dilated cardiomyopathy in patients with hypoadrenocorticism. Two dogs were presented to a single referral center for evaluation of weakness or collapse and were subsequently diagnosed with hypoadrenocorticism after a full diagnostic workup. Following the diagnosis of hypoadrenocorticism and administration of glucocorticoids and desoxycorticosterone pivalate, both dogs developed left-sided congestive heart failure and had systolic dysfunction diagnosed by echocardiogram. Both dogs were euthanized; one because of recurrent congestive heart failure and another because of a concern for poor long-term prognosis and decreased quality of life. The purpose of this case report is to document multiple cases of hypoadrenocorticism-associated systolic dysfunction and subsequent cardiogenic pulmonary edema in dogs.

Peptidoglycomics reveals compositional changes in peptidoglycan between biofilm- and planktonic-derived Pseudomonas aeruginosa.

Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating ß-1,4-linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured Pseudomonas aeruginosa We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de-N-acetylated muropeptides from P. aeruginosa The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.

Evolutionarily conserved Tbx5-Wnt2/2b pathway orchestrates cardiopulmonary development.

Codevelopment of the lungs and heart underlies key evolutionary innovations in the transition to terrestrial life. Cardiac specializations that support pulmonary circulation, including the atrial septum, are generated by second heart field (SHF) cardiopulmonary progenitors (CPPs). It has been presumed that transcription factors required in the SHF for cardiac septation, e.g., Tbx5, directly drive a cardiac morphogenesis gene-regulatory network. Here, we report instead that TBX5 directly drives Wnt ligands to initiate a bidirectional signaling loop between cardiopulmonary mesoderm and the foregut endoderm for endodermal pulmonary specification and, subsequently, atrial septation. We show that Tbx5 is required for pulmonary specification in mice and amphibians but not for swim bladder development in zebrafish. TBX5 is non-cell-autonomously required for pulmonary endoderm specification by directly driving Wnt2 and Wnt2b expression in cardiopulmonary mesoderm. TBX5 ChIP-sequencing identified cis-regulatory elements at Wnt2 sufficient for endogenous Wnt2 expression domains in vivo and required for Wnt2 expression in precardiac mesoderm in vitro. Tbx5 cooperated with Shh signaling to drive Wnt2b expression for lung morphogenesis. Tbx5 haploinsufficiency in mice, a model of Holt-Oram syndrome, caused a quantitative decrement of mesodermal-to-endodermal Wnt signaling and subsequent endodermal-to-mesodermal Shh signaling required for cardiac morphogenesis. Thus, Tbx5 initiates a mesoderm-endoderm-mesoderm signaling loop in lunged vertebrates that provides a molecular basis for the coevolution of pulmonary and cardiac structures required for terrestrial life.

Magnetic Entropy as a Proposed Gating Mechanism for Magnetogenetic Ion Channels.

Magnetically sensitive ion channels would allow researchers to better study how specific brain cells affect behavior in freely moving animals; however, recent reports of "magnetogenetic" ion channels based on biogenic ferritin nanoparticles have been questioned because known biophysical mechanisms cannot explain experimental observations. Here, we reproduce a weak magnetically mediated calcium response in HEK cells expressing a previously published TRPV4-ferritin fusion protein. We find that this magnetic sensitivity is attenuated when we reduce the temperature sensitivity of the channel but not when we reduce the mechanical sensitivity of the channel, suggesting that the magnetic sensitivity of this channel is thermally mediated. As a potential mechanism for this thermally mediated magnetic response, we propose that changes in the magnetic entropy of the ferritin particle can generate heat via the magnetocaloric effect and consequently gate the associated temperature-sensitive ion channel. Unlike other forms of magnetic heating, the magnetocaloric mechanism can cool magnetic particles during demagnetization. To test this prediction, we constructed a magnetogenetic channel based on the cold-sensitive TRPM8 channel. Our observation of a magnetic response in cold-gated channels is consistent with the magnetocaloric hypothesis. Together, these new data and our proposed mechanism of action provide additional resources for understanding how ion channels could be activated by low-frequency magnetic fields.

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.

FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates Its Delivery to the Vacuolar Degradation Pathway.

Recently, we described the ubiquitylation of PYL4 and PYR1 by the RING E3 ubiquitin ligase RSL1 at the plasma membrane of Arabidopsis thaliana This suggested that ubiquitylated abscisic acid (ABA) receptors might be targeted to the vacuolar degradation pathway because such ubiquitylation is usually an internalization signal for the endocytic route. Here, we show that FYVE1 (previously termed FREE1), a recently described component of the endosomal sorting complex required for transport (ESCRT) machinery, interacted with RSL1-receptor complexes and recruited PYL4 to endosomal compartments. Although the ESCRT pathway has been assumed to be reserved for integral membrane proteins, we show the involvement of this pathway in the degradation of ABA receptors, which can be associated with membranes but are not integral membrane proteins. Knockdown fyve1 alleles are hypersensitive to ABA, illustrating the biological relevance of the ESCRT pathway for the modulation of ABA signaling. In addition, fyve1 mutants are impaired in the targeting of ABA receptors for vacuolar degradation, leading to increased accumulation of PYL4 and an enhanced response to ABA Pharmacological and genetic approaches revealed a dynamic turnover of ABA receptors from the plasma membrane to the endosomal/vacuolar degradation pathway, which was mediated by FYVE1 and was dependent on RSL1. This process involves clathrin-mediated endocytosis and trafficking of PYL4 through the ESCRT pathway, which helps to regulate the turnover of ABA receptors and attenuate ABA signaling.

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 µM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKß are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS.

Defining the conditional basis of silicon phthalocyanine near-IR ligand exchange.

Bond cleavage reactions initiated by long-wavelength light are needed to extend the scope of the caged-uncaged paradigm into complex physiological settings. Axially unsymmetrical silicon phthalocyanines (SiPcs) undergo efficient release of phenol ligands in a reaction contingent on three factors - near-IR light (690 nm), hypoxia, and a thiol reductant. These studies detail efforts to define the mechanistic basis for this unique conditionally-dependent bond cleavage reaction. Spectroscopic studies provide evidence for the formation of a key phthalocyanine radical anion intermediate formed from the triplet state in a reductant-dependent manner. Computational chemistry studies indicate that phenol ligand solvolysis proceeds through a heptacoordinate silicon transition state and that this solvolytic process is favored following SiPc radical anion formation. These results provide insight regarding the central role that radical anion intermediates formed through photoinduced electron transfer with biological reductants can play in long-wavelength uncaging reactions.

VEGF and IGF Delivered from Alginate Hydrogels Promote Stable Perfusion Recovery in Ischemic Hind Limbs of Aged Mice and Young Rabbits.

Biomaterial-based delivery of angiogenic growth factors restores perfusion more effectively than bolus delivery methods in rodent models of peripheral vascular disease, but the same success has not yet been demonstrated in clinically relevant studies of aged or large animals. These studies explore, in clinically relevant models, a therapeutic angiogenesis strategy for the treatment of peripheral vascular disease that overcomes the challenges encountered in previous clinical trials. Alginate hydrogels providing sustained release of vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF) were injected into ischemic hind limbs in middle-aged and old mice, and also in young rabbits, as a test of the scalability of this local growth factor treatment. Spontaneous perfusion recovery diminished with increasing age, and only the combination of VEGF and IGF delivery from gels significantly rescued perfusion in middle-aged (13 months) and old (20 months) mice. In rabbits, the delivery of VEGF alone or in combination with IGF from alginate hydrogels, at a dose 2 orders of magnitude lower than the typical doses used in past rabbit studies, enhanced perfusion recovery when given immediately after surgery, or as a treatment for chronic ischemia. Capillary density measurements and angiographic analysis demonstrated the benefit of gel delivery. These data together suggest that alginate hydrogels providing local delivery of low doses of VEGF and IGF constitute a safe and effective treatment for hind-limb ischemia in clinically relevant animal models, thereby supporting the potential clinical translation of this concept.

The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.

Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca2+-dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser11 in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca2+-dependent Ser11 phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca2+ ions [K0.5(Ca2+) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser11 phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser451, RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca2+-signaling and the control of photosynthate partitioning during COS development.

Five-Month-Old Infants Have General Knowledge of How Nonsolid Substances Behave and Interact.

Experience puts people in touch with nonsolid substances, such as water, blood, and milk, which are crucial to survival. People must be able to understand the behavior of these substances and to differentiate their properties from those of solid objects. We investigated whether infants represent nonsolid substances as a conceptual category distinct from solid objects on the basis of differences in cohesiveness. Experiment 1 established that infants can distinguish water from a perceptually matched solid and can correctly predict whether the item will pass through or be trapped by a grid. Experiments 2 and 3 showed that infants extend this knowledge to less familiar granular substances. These experiments indicate that concepts of cohesive and noncohesive material appear early in development, apply across several types of nonsolid substances, and may serve as the basis of later knowledge of physical phases.

Human Papillomavirus Type 16 L2 DNA Methylation in Exfoliated Cervical Cells From College-Age Women.

OBJECTIVES: The Carolina Women's Care Study (CWCS) at the University of South Carolina followed 467 young women with the goal of identifying biomarkers of human papillomavirus (HPV) persistence. In this study, we analyzed the methylation of HPV16 DNA. METHODS: The aims of this study were to determine the methylation status of the HPV16 L2 gene in DNA isolated from exfoliated cervical cells collected longitudinally as part of the CWCS and to determine the prevalence of polymorphisms (single nucleotide polymorphisms [SNPs]) in folate metabolizing enzymes and DNA repair enzymes known to affect DNA methylation in blood-derived genomic DNA from CWCS participants. For methylation studies, DNA samples were bisulfite converted and amplified with the EpiTect Whole Bisulfitome kit. Polymerase chain reaction was performed for amplicons containing 5 CpG sites in L2. Pyrosequencing was carried out using EpigenDx and analyzed with PyroMark Software. Taqman genotyping assays were performed to determine selected SNP alleles in the CWCS cohort. RESULTS AND CONCLUSIONS: Methylation data were obtained for 82 samples from 27 participants. Of these, 22 participants were positive for HPV16 for 3 or more visits (≥12 months). Methylation in L2 was detectable, but methylation levels varied and were not associated with HPV16 persistence. No linearity of methylation levels over time was observed in participants for whom longitudinal data could be analyzed. Analysis of 9 selected SNPs did not reveal an association with persistence. We conclude that at early stages of infection methylation of HPV16 L2 DNA in Pap test samples is not a predictive biomarker of HPV persistence.