The Forkhead Transcription Factor Foxi1 Is a Master Regulator of Vacuolar H+-ATPase Proton Pump Subunits in the Inner Ear, Kidney and Epididymis

The vacuolar H+-ATPase dependent transport of protons across cytoplasmic membranes in FORE (forkhead related) cells of endolymphatic epithelium in the inner ear, intercalated cells of collecting ducts in the kidney and in narrow and clear cells of epididymis require expression of several subunits that assemble into a functional multimeric proton pump. We demonstrate that expression of four such subunits A1, B1, E2 and a4 all co-localize with the forkhead transcription factor Foxi1 in a subset of epithelial cells at these three locations. In cells, of such epithelia, that lack Foxi1 we fail to identify any expression of A1, B1, E2 and a4 demonstrating an important role for the transcription factor Foxi1 in regulating subunit availability. Promoter reporter experiments, electrophoretic mobility shift assays (EMSA) and site directed mutagenesis demonstrate that a Foxi1 expression vector can trans-activate an a4-promoter reporter construct in a dose dependent manner. Furthermore, we demonstrate using chromatin immunoprecipitation (ChIP) assays that Foxi1-dependent activation to a large extent depends on cis-elements at position −561/−547 in the a4 promoter. Thus, we provide evidence that Foxi1 is necessary for expression of at least four subunits in three different epithelia and most likely is a major determinant for proper assembly of a functional vacuolar H+-ATPase complex at these locations

Foxi1, an important gene for hearing, kidney function and male fertility

Aim: In this study we focused on the biological importance of the forkhead transcription factor Foxi1. The overall aim was to elucidate the functional role of Foxi1 in the inner ear, kidney and epididymis. When mice lacking the winged helix transcription factor Foxi1 were compared to their wt littermates in their response to acidic load, we discovered that Foxi1 deficient mice develop distal renal tubular acidosis (dRTA). Moreover, during breeding we observed the inability of Foxi1-/- males to generate offspring, indicating male infertility. In the light of these observations, together with our previous results from the inner ear of Foxi1-/- mice, we set out to examine the biological importance of Foxi1 in the inner ear, kidney and the epididymis. In this thesis we assess the expression pattern of Foxi1 and its potential downstream target genes in these three different tissues by using Northern blot analyses, in situ hybridization and immunohistochemistry. We demonstrated that Foxi1 is exclusively expressed in cells positive for the “kidney specific” vATPase subunits B1and a4, as well as the anion exchangers AE1 and pendrin, all characteristics for acid/base regulating cells of the inner ear, kidney and epididymis, called “mitochondria rich” cells. We present evidence that Foxi1 is required for the expression of these genes. Furthermore, in transfection experiments and in electrophoretic mobility shift assays (EMSA) we established direct transcriptional activation of promoter constructs by Foxi1. This activation was abolished when specific Foxi1 cis-elements on the promoters were mutated. Previously we have shown that mice with target disruption of the Foxi1 locus exhibit severe cochlear and vestibular malformation and that Foxi1 is expressed in endolymphatic epithelial cells important for regulation of volume and composition of endolymph fluid. We have also shown that pendrin is missing from endolymphatic epithelium in Foxi1 deficient mice, making human FOXI1 a potential transcriptional activator of the gene coding for pendrin, the SLC26A4 gene. While recessive mutations in the SLC26A4 gene are known to be responsible for Pendred syndrome (PS) and non-syndromic hearing loss associated with enlarged vestibular aqueduct (EVA), a large percentage of patients with this phenotype lack mutations in the SLC26A4 coding region in one or both alleles. We have identified and characterized a key transcriptional regulatory element in the SLC26A4 promoter that binds FOXI1. Moreover, we have identified six PS or non-syndromic EVA patients with mutations in FOXI1 that inhibits its activation of SLC26A4 transcription In summary, we present molecular data showing that Foxi1 is required for the expression of some of the most important transporter molecules of acid-base regulation in the inner ear, kidney and epididymis, establishing Foxi1 as an essential regulator of fluid homeostasis in these organs. Hence, mice deficient for Foxi1, beside their early onset deafness, develop dRTA and male infertility. We also demonstrate that mutations in the human FOXI1 gene are involved in pathogenetic mechanisms underlying human deafness. Finally, these results together with our earlier findings in mice support the hypothesis that mutations in the human FOXI1 gene might prove to cause a sensorineural deafness syndrome with distal renal tubular acidosis and male infertility

EFFICIENCY OF DIFFERENT CARBON SOURCES DURING SINTERING OF CR-PREALLOYED PM STEELS: GRAPHENE, GRAPHITE OR CARBON BLACK?

Prerequisite for development of inter-particle necks as well as carbon dissolution in the steel matrix is efficient removal of the iron oxide layer, covering surface of the base steel powder. Reactivity of admixed carbon source strongly affects efficiency of the surface oxide removal and so determines inter-particle necks development, further carbon dissolution and connected to this microstructure development. The present study is focused on the critical evaluation of the parameters, determining chemical reactivity of different carbon sources. Effect of admixing of number of different carbon sources – graphene nanoplatelets, synthetic and natural graphite grades with different particle size as well as carbon black powders – on the sintering behaviour of Cr-prealloyed powder was evaluated. Deoxidation, microstructure and inter-particle necks development were studied by means of metallography and fractography. Carbon source structure, powder size and interatomic bond characteristics were established to be the most important properties determining carbon reactivity

EFFICIENCY OF DIFFERENT CARBON SOURCES DURING SINTERING OF CR-PREALLOYED PM STEELS: GRAPHENE, GRAPHITE OR CARBON BLACK?

Prerequisite for development of inter-particle necks as well as carbon dissolution in the steel matrix is efficient removal of the iron oxide layer, covering surface of the base steel powder. Reactivity of admixed carbon source strongly affects efficiency of the surface oxide removal and so determines inter-particle necks development, further carbon dissolution and connected to this microstructure development. The present study is focused on the critical evaluation of the parameters, determining chemical reactivity of different carbon sources. Effect of admixing of number of different carbon sources – graphene nanoplatelets, synthetic and natural graphite grades with different particle size as well as carbon black powders – on the sintering behaviour of Cr-prealloyed powder was evaluated. Deoxidation, microstructure and inter-particle necks development were studied by means of metallography and fractography. Carbon source structure, powder size and interatomic bond characteristics were established to be the most important properties determining carbon reactivity

Additive Manufacturing of MnAl(C)-Magnets

Permanent magnets are becoming more and more relevant for modern society. As the most widely used permanent magnets contain rare-earth elements, the increased dependence on these strategic elements is worrisome, and the pursuit for rare-earth free alternatives has become a strategic goal in many countries. The metastable and ferromagnetic τ-phase that forms in the MnAl(C) system is one of the most promising alternatives, and since its discovery, major efforts have been made to improve its performance and realize its full potential. One major factor that has prevented a widespread commercialization of MnAl(C) permanent magnets is their relatively low coercivity. Here, we demonstrate that additive manufacturing, using laser powder bed fusion, can be used to produce MnAl in its high-temperature polymorph (ε, hcp), which can be subsequently transformed, through post-heat treatments to the ferromagnetic τ-phase. Although we successfully obtained a preferential orientation of the ε-phase with parallel to the build direction, this did not translate into a strong preferential orientation in the τ-phase, thus indicating that the phase transformation occurs by the migration of incoherent interfaces. The MnAl(C) samples are characterized by a density of ≈4.4 g/cm3, a saturation magnetization of 39.3 Am2/kg, a coercivity of 168 kA/m, and a remanence of 17.5 Am2/kg

Cytogenetic changes in nonmalignant breast tissue

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldCytogenetic changes are common in breast cancer and have also been described in fibroadenomas and fibrocystic disease, but not in histologically normal breast tissue. Cytogenetic analysis was performed on nonmalignant breast tissue from benign breast lumps (n = 8), reduction mammoplasties (n = 31), and grossly nontumorous tissue from cancerous breasts (n = 84), using standard techniques and G-banding. All samples were reviewed histologically. Clonal chromosomal changes were found in three of eight benign breast tumors (38%). Of the reduction mammoplasties, 17 samples contained nonproliferative changes, and three of these (18%) showed a clonal deletion of 3p. No pathology was identified in the other 14 samples, of which one (7%) contained two clonal changes, apparently balanced translocations. Of nontumorous tissues from cancerous breasts, 15 (18%) showed clonal chromosomal abnormalities. Five of these samples were histologically normal. Two clones were identical to those found in the corresponding cancer. In 18 additional samples, single cells were detected with the same change as that seen in clones or single cells in the cancer. Only 4 of these 20 samples contained detectable cancer cells. Clonal abnormalities found in two or more samples included trisomies X, 7, and 20 and monosomies 19 and 18. Clonal changes were not significantly more frequent in proliferative than in nonproliferative lesions. The Icelandic BRCA2 founder mutation, 999del5, was detected in four samples, all histologically normal, two of which had clonal chromosomal abnormalities. In conclusion, clonal chromosomal changes are not infrequent in nonmalignant breast tissue and can be detected even in the absence of histological abnormalities

Niobium carbide – Nickel-niobium alloy composites from a nickel coated powder: Microstructural development during sintering

The sintering of a 14 ​wt% nickel nano-dot coated NbC powder was investigated using dilatometry in combination with XRD, ToF-ERDA, PIXE, SEM-EDS/EBSD/TKD, and TEM-HAADF/EDS/EELS for analysis of samples heated to 1375, 1405 and 1500 ​°C, with a 30 ​min annealing. The main sintering step at 1110–1375 ​°C, was succeeded by a slower step, centred at 1405 ​°C, before the final smaller densification step to 1500 ​°C. NbC grain-growth took place on heating at 1500 ​°C for 30 ​min; from ca. 0.5–5 ​μm (average 1 ​μm) at 1375 and 1405 ​°C to ca. 5–30 ​μm (average 10 ​μm) 1500 ​°C for 30 ​min. The NbC0.91-0.94 phase showed a micro-hardness of 1700–2300 HV0.05. The binder phase consisted of the unprecedented textured c-Nb0.15Ni0.85 and h-Nb0.2Ni0.8 phases. The Nb–Ni binder phase yielded micro-hardness values of 1150–1250 (1375–1405 ​°C) to 810 HV0.05 (1500 ​°C, 30 ​min), which greatly exceeds nickel and is higher than Fe3Al

Transcriptional Control of SLC26A4 Is Involved in Pendred Syndrome and Nonsyndromic Enlargement of Vestibular Aqueduct (DFNB4)

Although recessive mutations in the anion transporter gene SLC26A4 are known to be responsible for Pendred syndrome (PS) and nonsyndromic hearing loss associated with enlarged vestibular aqueduct (EVA), also known as “DFNB4,” a large percentage of patients with this phenotype lack mutations in the SLC26A4 coding region in one or both alleles. We have identified and characterized a key transcriptional regulatory element in the SLC26A4 promoter that binds FOXI1, a transcriptional activator of SLC26A4. In nine patients with PS or nonsyndromic EVA, a novel c.−103T→C mutation in this regulatory element interferes with FOXI1 binding and completely abolishes FOXI1-mediated transcriptional activation. We have also identified six patients with mutations in FOXI1 that compromise its ability to activate SLC26A4 transcription. In one family, the EVA phenotype segregates in a double-heterozygous mode in the affected individual who carries single mutations in both SLC26A4 and FOXI1. This finding is consistent with our observation that EVA occurs in the Slc26a4(+/−); Foxi1(+/−) double-heterozygous mouse mutant. These results support a novel dosage-dependent model for the molecular pathogenesis of PS and nonsyndromic EVA that involves SLC26A4 and its transcriptional regulatory machinery

To Alfred Deakin

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldIt has been proposed that hypoxia favors the growth of tumor cells over normal cells, particularly tumor cells carrying TP53 mutations. Cytogenetic studies of breast cancer have shown that highly complex karyotypes seen in direct harvest preparations are rarely detected after short-term culture. In this study, 34 paired samples of breast carcinomas and grossly nontumorous tissue from the same breast were cultured at 20 and 5% (12 samples) or 20 and 0% oxygen (22 samples). Both carcinoma samples and nontumorous tissue survived at 0% oxygen. Recovery for 24 hours at 20% produced good yields for cytogenetic analysis. Lower oxygen levels did not specifically stimulate growth of tumor cells. Samples with TP53 mutations showed a consistently increased growth under anaerobic hypoxic conditions. Culture at 5% oxygen did not generally reveal more karyotypic abnormalities than found at 20%. In the samples cultured at 0 and 20%, karyotypic abnormalities were detected only in anaerobic hypoxic culture in two cases. Of the only four samples where more complex karyotypes were detected in the low-oxygen culture, two were TP53 mutated. Hypoxic treatment followed by recovery at 20% oxygen may thus increase the yield of complex karyotypes from a subset of breast carcinomas, particularly those with mutated TP53

Development of permanent magnet MnAlC/polymer composites and flexible filament for bonding and 3D-printing technologies

Searching for high-performance permanent magnets components with no limitation in shape and dimensions is highly desired to overcome the present design and manufacturing restrictions, which affect the efficiency of the final devices in energy, automotive and aerospace sectors. Advanced 3D-printing of composite materials and related technologies is an incipient route to achieve functional structures avoiding the limitations of traditional manufacturing. Gas-atomized MnAlC particles combined with polymer have been used in this work for fabricating scalable rare earth-free permanent magnet composites and extruded flexible filaments with continuous length exceeding 10 m. Solution casting has been used to synthesize homogeneous composites with tuned particles content, made of a polyethylene (PE) matrix embedding quasi-spherical particles of the ferromagnetic τ-MnAlC phase. A maximum filling factor of 86.5 and 72.3% has been obtained for the composite and the filament after extrusion, respectively. The magnetic measurements reveal no deterioration of the properties of the MnAlC particles after the composite synthesis and filament extrusion. The produced MnAlC/PE materials will serve as precursors for an efficient and scalable design and fabrication of end-products by different processing techniques (polymerized cold-compacted magnets and 3D-printing, respectively) in view of technological applications (from micro electromechanical systems to energy and transport applications)