Quantification of FAM20A in human milk and identification of calcium metabolism proteins

BACKGROUND: FAM20A, a recently discovered protein, is thought to have a fundamental role in inhibiting ectopic calcification. Several studies have demonstrated that variants of FAM20A are causative for the rare autosomal recessive disorder, enamel-renal syndrome (ERS). ERS is characterized by defective mineralization of dental enamel and nephrocalcinosis suggesting that FAM20A is an extracellular matrix protein, dysfunction of which causes calcification of the secretory epithelial tissues. FAM20A is a low-abundant protein that is difficult to detect in biofluids such as blood, saliva, and urine. Thus, we speculated the abundance of FAM20A to be high in human milk, since the secretory epithelium of lactating mammary tissue is involved in the secretion of highly concentrated calcium. Therefore, the primary aim of this research is to describe the processes/methodology taken to quantify FAM20A in human milk and identify other proteins involved in calcium metabolism. METHOD: This study used mass spectrometry-driven quantitative proteomics: (1) to quantify FAM20A in human milk of three women and (2) to identify proteins associated with calcium regulation by bioinformatic analyses on whole and milk fat globule membrane fractions. RESULTS: Shotgun MS/MS driven proteomics identified FAM20A in whole milk, and subsequent analysis using targeted proteomics also successfully quantified FAM20A in all samples. Combination of sample preparation, fractionation, and LC-MS/MS proteomics analysis generated 136 proteins previously undiscovered in human milk; 21 of these appear to be associated with calcium metabolism. CONCLUSION: Using mass spectrometry-driven proteomics, we successfully quantified FAM20A from transitional to mature milk and obtained a list of proteins involved in calcium metabolism. Furthermore, we show the value of using a combination of both shotgun and targeted driven proteomics for the identification of this low abundant protein in human milk

A new experimental snow avalanche test site at Seehore peak in Aosta Valley (NW Italian Alps) - Part II: Engineering aspects

The estimate of the effects produced by the impact of a snow avalanche against an obstacle is of the utmost importance in designing safe mountain constructions. For this purpose, an ad-hoc instrumented obstacle was designed and built in order to measure impact forces of small and medium snow avalanches at Seehore peak (NW Italian Alps). The structural design had to consider several specific and unusual demands dictated by the difficult environment. In this article, the new test facility is described from the engineering point of view, discussing the most important aspects of the analyzed problems which were solved before and after the construction. The performance of the instrumented obstacle in the first two operating seasons, and some proposals for future upgrading are eventually illustrate

Anomalies and WZW-term of two-flavour QCD

The U(2)_R x U(2)_L symmetry of QCD with two massless flavours is subject to anomalies which affect correlation functions involving the singlet currents A^0_\mu or V^0_\mu. These are relevant for pion-photon interactions, because - for two flavours - the electromagnetic current contains a singlet piece. We give the effective Lagrangian required for the corresponding low energy analysis to next-to-leading order, without invoking an expansion in the mass of the strange quark. In particular, the Wess-Zumino-Witten term that accounts for the two-flavour anomalies within the effective theory is written down in closed form.Comment: 17 pages, 1 figur

Recent Developments in Chiral Perturbation Theory

I review recent developments in chiral perturbation theory (CHPT) which is the effective field theory of the standard model below the chiral symmetry breaking scale. The effective chiral Lagrangian formulated in terms of the pseudoscalar Goldstone bosons ($\pi, \, K, \, \eta$) is briefly discussed. It is shown how one can gain insight into the ratios of the light quark masses and to what extent these statements are model--independent. A few selected topics concerning the dynamics and interactions of the Goldstone bosons are considered. These are $\pi \pi$ and $\pi K$ scattering, some non--leptonic kaon decays and the problem of strong pionic final state interactions. CHPT also allows to make precise statements about the temperature dependence of QCD Green functions and the finite size effects related to the propagation of the (almost) massless pseudoscalar mesons. A central topic is the inclusion of matter fields, baryon CHPT. The relativistic and the heavy fermion formulation of coupling the baryons to the Goldstone fields are discussed. As applications, photo--nucleon processes, the $\pi N$ $\Sigma$--term and non--leptonic hyperon decays are presented. Implications of the spontaneously broken chiral symmetry on the nuclear forces and meson exchange currents are also described. Finally, the use of effective field theory methods in the strongly coupled Higgs sector and in the calculation of oblique electroweak corrections is touched upon.Comment: TeX, 110 pages, 15 figures available upon request, BUTP-93/0

Mir-132/212 is required for maturation of binocular matching of orientation preference and depth perception

MicroRNAs (miRNAs) are known to mediate post-transcriptional gene regulation, but their role in postnatal brain development is still poorly explored. We show that the expression of many miRNAs is dramatically regulated during functional maturation of the mouse visual cortex with miR-132/212 family being one of the top upregulated miRNAs. Age-downregulated transcripts are significantly enriched in miR-132/miR-212 putative targets and in genes upregulated in miR-132/212 null mice. At a functional level, miR-132/212 deletion affects development of receptive fields of cortical neurons determining a specific impairment of binocular matching of orientation preference, but leaving orientation and direction selectivity unaltered. This deficit is associated with reduced depth perception in the visual cliff test. Deletion of miR-132/212 from forebrain excitatory neurons replicates the binocular matching deficits. Thus, miR-132/212 family shapes the age-dependent transcriptome of the visual cortex during a specific developmental window resulting in maturation of binocular cortical cells and depth perception

The genetic architecture of membranous nephropathy and its potential to improve non-invasive diagnosis

Membranous Nephropathy (MN) is a rare autoimmune cause of kidney failure. Here we report a genome-wide association study (GWAS) for primary MN in 3,782 cases and 9,038 controls of East Asian and European ancestries. We discover two previously unreported loci, NFKB1 (rs230540, OR = 1.25, P = 3.4 × 10-12) and IRF4 (rs9405192, OR = 1.29, P = 1.4 × 10-14), fine-map the PLA2R1 locus (rs17831251, OR = 2.25, P = 4.7 × 10-103) and report ancestry-specific effects of three classical HLA alleles: DRB1*1501 in East Asians (OR = 3.81, P = 2.0 × 10-49), DQA1*0501 in Europeans (OR = 2.88, P = 5.7 × 10-93), and DRB1*0301 in both ethnicities (OR = 3.50, P = 9.2 × 10-23 and OR = 3.39, P = 5.2 × 10-82, respectively). GWAS loci explain 32% of disease risk in East Asians and 25% in Europeans, and correctly re-classify 20-37% of the cases in validation cohorts that are antibody-negative by the serum anti-PLA2R ELISA diagnostic test. Our findings highlight an unusual genetic architecture of MN, with four loci and their interactions accounting for nearly one-third of the disease risk

Polycystic kidney disease with hyperinsulinemic hypoglycemia caused by a promoter mutation in PMM2

Hyperinsulinemic hypoglycemia (HI) and congenital polycystic kidney disease (PKD) are rare, genetically heterogeneous disorders. The co-occurrence of these disorders (HIPKD) in 17 children from 11 unrelated families suggested an unrecognized genetic disorder. Whole-genome linkage analysis in five informative families identified a single significant locus on chromosome 16p13.2 (logarithm of odds score 6.5). Sequencing of the coding regions of all linked genes failed to identify biallelic mutations. Instead, we found in all patients a promoter mutation (c.-167G>T) in the phosphomannomutase 2 gene (PMM2), either homozygous or in trans with PMM2 coding mutations. PMM2 encodes a key enzyme in N-glycosylation. Abnormal glycosylation has been associated with PKD, and we found that deglycosylation in cultured pancreatic β cells altered insulin secretion. Recessive coding mutations in PMM2 cause congenital disorder of glycosylation type 1a (CDG1A), a devastating multisystem disorder with prominent neurologic involvement. Yet our patients did not exhibit the typical clinical or diagnostic features of CDG1A. In vitro, the PMM2 promoter mutation associated with decreased transcriptional activity in patient kidney cells and impaired binding of the transcription factor ZNF143. In silico analysis suggested an important role of ZNF143 for the formation of a chromatin loop including PMM2. We propose that the PMM2 promoter mutation alters tissue-specific chromatin loop formation, with consequent organ-specific deficiency of PMM2 leading to the restricted phenotype of HIPKD. Our findings extend the spectrum of genetic causes for both HI and PKD and provide insights into gene regulation and PMM2 pleiotropy