Functional dissection of the Drosophila Kallmann's syndrome protein DmKal-1

BACKGROUND: Anosmin-1, the protein implicated in the X-linked Kallmann's syndrome, plays a role in axon outgrowth and branching but also in epithelial morphogenesis. The molecular mechanism of its action is, however, widely unknown. Anosmin-1 is an extracellular protein which contains a cysteine-rich region, a whey acidic protein (WAP) domain homologous to some serine protease inhibitors, and four fibronectin-like type III (FnIII) repeats. Drosophila melanogaster Kal-1 (DmKal-1) has the same protein structure with minor differences, the most important of which is the presence of only two FnIII repeats and a C-terminal region showing a low similarity with the third and the fourth human FnIII repeats. We present a structure-function analysis of the different DmKal-1 domains, including a predicted heparan-sulfate binding site. RESULTS: This study was performed overexpressing wild type DmKal-1 and a series of deletion and point mutation proteins in two different tissues: the cephalopharyngeal skeleton of the embryo and the wing disc. The overexpression of DmKal-1 in the cephalopharyngeal skeleton induced dosage-sensitive structural defects, and we used these phenotypes to perform a structure-function dissection of the protein domains. The reproduction of two deletions found in Kallmann's Syndrome patients determined a complete loss of function, whereas point mutations induced only minor alterations in the activity of the protein. Overexpression of the mutant proteins in the wing disc reveals that the functional relevance of the different DmKal-1 domains is dependent on the extracellular context. CONCLUSION: We suggest that the role played by the various protein domains differs in different extracellular contexts. This might explain why the same mutation analyzed in different tissues or in different cell culture lines often gives opposite phenotypes. These analyses also suggest that the FnIII repeats have a main and specific role, while the WAP domain might have only a modulator role, strictly connected to that of the fibronectins

Measurement of the Bottom-Strange Meson Mixing Phase in the Full CDF Data Set

We report a measurement of the bottom-strange meson mixing phase \beta_s using the time evolution of B0_s -> J/\psi (->\mu+\mu-) \phi (-> K+ K-) decays in which the quark-flavor content of the bottom-strange meson is identified at production. This measurement uses the full data set of proton-antiproton collisions at sqrt(s)= 1.96 TeV collected by the Collider Detector experiment at the Fermilab Tevatron, corresponding to 9.6 fb-1 of integrated luminosity. We report confidence regions in the two-dimensional space of \beta_s and the B0_s decay-width difference \Delta\Gamma_s, and measure \beta_s in [-\pi/2, -1.51] U [-0.06, 0.30] U [1.26, \pi/2] at the 68% confidence level, in agreement with the standard model expectation. Assuming the standard model value of \beta_s, we also determine \Delta\Gamma_s = 0.068 +- 0.026 (stat) +- 0.009 (syst) ps-1 and the mean B0_s lifetime, \tau_s = 1.528 +- 0.019 (stat) +- 0.009 (syst) ps, which are consistent and competitive with determinations by other experiments.Comment: 8 pages, 2 figures, Phys. Rev. Lett 109, 171802 (2012

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Renal gluconeogenesis: an underestimated role of the kidney in systemic glucose metabolism

Glucose levels are tightly regulated at all times. Gluconeogenesis is the metabolic pathway dedicated to glucose synthesis from non-hexose precursors. Gluconeogenesis is critical for glucose homoeostasis, particularly during fasting or stress conditions. The renal contribution to systemic gluconeogenesis is increasingly recognized. During the post-absorptive phase, the kidney accounts for ∼40% of endogenous gluconeogenesis, occurring mainly in the kidney proximal tubule. The main substrate for renal gluconeogenesis is lactate and the process is regulated by insulin and cellular glucose levels, but also by acidosis and stress hormones. The kidney thus plays an important role in the maintenance of glucose and lactate homoeostasis during stress conditions. The impact of acute and chronic kidney disease and proximal tubular injury on gluconeogenesis is not well studied. Recent evidence shows that in both experimental and clinical acute kidney injury, impaired renal gluconeogenesis could significantly participate in systemic metabolic disturbance and thus alter the prognosis. This review summarizes the biochemistry of gluconeogenesis, the current knowledge of kidney gluconeogenesis, its modifications in kidney disease and the clinical relevance of this fundamental biological process in human biology

Postfertilization Autophagy of Sperm Organelles Prevents Paternal Mitochondrial DNA Transmission

International audienc

Spatiotemporal Landscape of Kidney Tubular Responses to Glomerular Proteinuria

BACKGROUND Large increases in glomerular protein filtration induce major changes in body homeostasis and increase risk of kidney functional decline and cardiovascular disease. We investigated how elevated protein exposure modifies the landscape of tubular function along the entire nephron, to understand the cellular changes that mediate these important clinical phenomena. METHODS We conducted single nuclei RNA sequencing, functional intravital imaging, and antibody staining to spatially map transport processes along the mouse kidney tubule. We then delineated how these were altered in a transgenic mouse model of inducible glomerular proteinuria (POD-ATTAC) at 7 and 28 days. RESULTS Glomerular proteinuria activated large-scale and pleotropic changes in gene expression in all major nephron sections. Extension of protein uptake from early (S1) to later (S2) parts of the proximal tubule initially triggered dramatic expansion of a hybrid S1/2 population, followed by injury and failed repair, with the cumulative effect of loss of canonical S2 functions. Proteinuria also induced acute injury in S3. Meanwhile, overflow of luminal proteins to the distal tubule caused transcriptional convergence between specialized regions and generalized dedifferentiation. CONCLUSIONS Proteinuria modulated cell signaling in tubular epithelia and causes distinct patterns of remodeling and injury in a segment specific manner

A transfer learning framework to elucidate the clinical relevance of altered proximal tubule cell states in kidney disease

Summary: The application of single-cell technologies in clinical nephrology remains elusive. We generated an atlas of transcriptionally defined cell types and cell states of human kidney disease by integrating single-cell signatures reported in the literature with newly generated signatures obtained from 5 patients with acute kidney injury. We used this information to develop kidney-specific cell-level information ExtractoR (K-CLIER), a transfer learning approach specifically tailored to evaluate the role of cell types/states on bulk RNAseq data. We validated the K-CLIER as a reliable computational framework to obtain a dimensionality reduction and to link clinical data with single-cell signatures. By applying K-CLIER on cohorts of patients with different kidney diseases, we identified the most relevant cell types associated with fibrosis and disease progression. This analysis highlighted the central role of altered proximal tubule cells in chronic kidney disease. Our study introduces a new strategy to exploit the power of single-cell technologies toward clinical applications

Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54

Hypogonadotropic hypogonadism is defined as a deficiency of the pituitary secretion of follicle-stimulating hormone and luteinizing hormone, which results in the impairment of pubertal maturation and of reproductive function. In the absence of pituitary or hypothalamic anatomical lesions and of anosmia (Kallmann syndrome), hypogonadotropic hypogonadism is referred to as isolated hypogonadotropic hypogonadism (IHH). A limited number of IHH cases are due to loss-of-function mutations of the gonadotropin-releasing hormone receptor. To identify additional gene defects leading to IHH, a large consanguineous family with five affected siblings and with a normal gonadotropin-releasing hormone receptor coding sequence was studied. Homozygosity whole-genome mapping allowed the localization of a new locus within the short arm of chromosome 19 (19p13). Sequencing of several genes localized within this region showed that all affected siblings of the family carried a homozygous deletion of 155 nucleotides in the GPR54 gene. This deletion encompassed the splicing acceptor site of intron 4–exon 5 junction and part of exon 5. The deletion was absent or present on only one allele in unaffected family members. GPR54 has been initially identified as an orphan G protein-coupled receptor with 40% homology to galanin receptors. Recently, a 54-aa peptide derived from the KiSS1 protein was identified as a ligand of GPR54. The present study shows that loss of function of GPR54 is a cause of IHH, and it identifies GPR54 and possibly KiSS1 protein-derived peptide as playing a major and previously unsuspected role in the physiology of the gonadotropic axis