Distinct regulatory elements of SLC6A14 expression contribute to modification of cystic fibrosis phenotypes

In mouse Paneth cells, alpha-defensins, termed cryptdins (Crps), are activated by matrix metalloproteinase-7-mediated proteolysis of inactive precursors (pro-Crps) to bactericidal forms. The activating cleavage step at Ser(43) downward arrow Ile(44) in mouse pro-Crp4-(20-92) removes nine acidic amino acids that collectively block the membrane-disruptive behavior of the Crp4 moiety of the proform. This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) are responsible for blocking bactericidal activity and membrane disruption. To test whether specific cluster(s) of electronegative amino acids in pro-Crp4-(20-43) have specific positional effects that block bactericidal peptide activity and membrane disruption, acidic residues positioned at the distal (Asp(20), Asp(26), Glu(27), and Glu(28)), mid (Glu(32) and Glu(33)), and proximal (Glu(37), Glu(38), and Asp(39)) clusters in pro-Crp4-(20-92) were mutagenized, and variants were assayed for differential effects of mutagenesis on bactericidal peptide activity. Substitution of the mid and proximal Asp and Glu clusters with Gly produced additive effects with respect to the induction of both bactericidal activity and membrane permeabilization of live Escherichia coli ML35 cells. In contrast, substitution of distal Glu and Asp residues with Gly or their deletion resulted in pro-Crp4-(20-92) variants with bactericidal and membrane-disruptive activities equal to or greater than that of fully mature Crp4. These findings support the conclusion that the most distal N-terminal anionic residues of pro-Crp4-(20-92) are primarily responsible for blocking Crp4-mediated membrane disruption in the precursor.

The semilethal skeletal malformation syndrome campomelic dysplasia (CD) with or without XY sex reversal is caused by mutations within the SOX9 gene on 17q24.3 or by chromosomal aberrations (translocations, inversions or deletions) with breakpoints outside the SOX9 coding region. The previously published CD translocation breakpoints upstream of SOX9 fall into two clusters: a proximal cluster with breakpoints between 50-300 kb and a distal cluster with breakpoints between 899-932 kb. Here, we present clinical, cytogenetic and molecular data from two novel CD translocation cases. Case 1 with karyotype 46,XY,t(1;17)(q42.1;q24.3) has characteristic symptoms of CD, including mild tibial bowing, cryptorchidism and hypospadias. By standard fluorescence in situ hybridization (FISH) and by high-resolution fiber FISH, the 17q breakpoint was mapped 375 kb from SOX9, defining the centromeric border of the proximal breakpoint cluster region. Case 2 with karyotype 46,X,t(Y;17)(q11.2;q24.3) has the acampomelic form of CD and complete XY sex reversal. By FISH and somatic cell hybrid analysis, the 17q breakpoint was mapped 789 kb from SOX9, defining the telomeric border of the distal breakpoint cluster region. We discuss the structure of the 1 Mb cis-control region upstream of SOX9 and the correlation between the position of the 14 mapped translocation breakpoints with respect to disease severity and XY sex reversal.

Cloning of crucian carp ( Carassius cuvieri) metallothionein-II gene and characterization of its gene promoter region

Two clusters of rat Nkrp1 genes can be distinguished based on phylogenetic relationships and functional characteristics. The proximal (centromeric) cluster encodes the well-studied NKR-P1A and NKR-P1B receptors and the distal cluster, the largely uncharacterized, NKR-P1F and NKR-P1G receptors. The inhibitory NKR-P1G receptor is expressed only by the Ly49s3+ NK cell subset as detected by RT-PCR, while the activating NKR-P1F receptor is detected in both Ly49s3+ and NKR-P1B+ NK cells. The mouse NKR-P1G ortholog is expressed by both NKR-P1D− and NKR-P1D+ NK cells in C57BL/6 mice. The rat and mouse NKR-P1F and NKR-P1G receptors demonstrate a striking, cross-species conservation of specificity for Clr ligands. NKR-P1F and NKR-P1G reporter cells reacted with overlapping panels of tumour cell lines and with cells transiently transfected with rat Clr2, Clr3, Clr4, Clr6 and Clr7 and mouse Clrc, Clrf, Clrg and Clrd/x, but not with Clr11 or Clrb, which serve as ligands for NKR-P1 from the proximal cluster. These data suggest that the conserved NKR-P1F and NKR-P1G receptors function as promiscuous receptors for a rapidly evolving family of Clr ligands in rodent NK cells.

Disease variation can be substantial even in conditions with a single gene etiology such as cystic fibrosis (CF). Simultaneously studying the effects of genes and environment may provide insight into the causes of variation. To determine whether secondhand smoke exposure is associated with lung function and other outcomes in individuals with CF, whether socioeconomic status affects the relationship between secondhand smoke exposure and lung disease severity, and whether specific gene-environment interactions influence the effect of secondhand smoke exposure on lung function. Retrospective assessment of lung function, stratified by environmental and genetic factors. Data were collected by the US Cystic Fibrosis Twin and Sibling Study with missing data supplemented by the Cystic Fibrosis Foundation Data Registry. All participants were diagnosed with CF, were recruited between October 2000 and October 2006, and were primarily from the United States. Disease-specific cross-sectional and longitudinal measures of lung function. Of 812 participants with data on secondhand smoke in the home, 188 (23.2%) were exposed. Of 780 participants with data on active maternal smoking during gestation, 129 (16.5%) were exposed. Secondhand smoke exposure in the home was associated with significantly lower cross-sectional (9.8 percentile point decrease; P < .001) and longitudinal lung function (6.1 percentile point decrease; P = .007) compared with those not exposed. Regression analysis demonstrated that socioeconomic status did not confound the adverse effect of secondhand smoke exposure on lung function. Interaction between gene variants and secondhand smoke exposure resulted in significant percentile point decreases in lung function, namely in CFTR non-DeltaF508 homozygotes (12.8 percentile point decrease; P = .001), TGFbeta1-509 TT homozygotes (22.7 percentile point decrease; P = .006), and TGFbeta1 codon 10 CC homozygotes (20.3 percentile point decrease; P = .005). Any exposure to secondhand smoke adversely affects both cross-sectional and longitudinal measures of lung function in individuals with CF. Variations in the gene that causes CF (CFTR) and a CF-modifier gene (TGFbeta1) amplify the negative effects of secondhand smoke exposure.

Role of SLC6A14 as a Modifier of Cystic Fibrosis Phenotype

The retroviral oncogene v-myb is a mutated and truncated version of the c-myb proto-oncogene and encodes a transcription factor (v-Myb) that specifically transforms myelomonocytic cells. Two different variants of v-myb, transduced independently by the oncogenic chicken retroviruses AMV and E26, have been characterized. It is believed that both variants of v-Myb transform myelomonocytic cells by affecting the expression of specific genes; however, no target genes common to both oncogenic viruses have been identified. Here, we describe the identification of a novel v-Myb target gene, designated as tom-1 (target of myb 1). The tom-1 gene has two promoters, one of which is Myb-inducible. tom-1 is expressed at elevated levels in AMV-transformed as well as in E26-transformed myeloid cells. We show that tom-1 activation by v-Myb does not require de novo protein synthesis and that the Myb-inducible tom-1 promoter contains a functional Myb binding site. Thus, tom-1 is the first example of a direct target gene for both oncogenic forms of the v-myb gene. Further analysis of the Myb-inducible tom-1 promoter shows that a C/EBP binding site is juxtaposed to the Myb binding site and that C/EBP is required for the Myb-dependent activation of the promoter. Together with previous work our results suggest that C/EBP may be a general cooperation partner for v-Myb in myelomonocytic cells.

The GAP-43 promoter region contains seven E-boxes (E1 to E7) that are organized in two clusters, a distal cluster (E3 to E7) and a proximal cluster (E1 and E2). Deletion analysis and site-directed mutagenesis of the GAP-43 promoter region showed that only the most proximal E1 E-box significantly modulates GAP-43 promoter activity. This E-box is conserved between the rat and human GAP-43 promoter sequences in terms of flanking sequence, core sequence (CAGTTG), and position. We found that endogenous E-box-binding proteins present in neuronal N18 cells recognize the E1 E-box and activate the GAP-43 promoter. The transcriptional activity of the GAP-43 promoter was repressed not only by the negative regulator Id2 protein, but also by two class A basic helix-loop-helix proteins, E12 and ME1a. In vitro analyses showed that both ME1a and E12 bind to the E1 E-box as homodimers. By Northern analyses, we established an inverse correlation between the level of E12 and ME1a mRNAs and GAP-43 mRNA in various neuronal cell lines as well as in ME1a-overexpressing PC12 cells. Therefore, we have identified a cis-acting element, the E1 E-box, located in the GAP-43 promoter region that modulates either positively or negatively the expression of the GAP-43 gene depending on which E-box-binding proteins occupy this site. Together, these data indicate that basic helix-loop-helix transcription factors regulate the expression of the GAP-43 gene and that the class A ME1a and E12 proteins act as down-regulators of GAP-43 expression.

Functional comparison of the metal-regulated transcriptional control regions of metallothionein genes from cadmium-sensitive and tolerant fish species

Metastasis is the main obstacle for the treatment of gastric cancer (GC), leading to low survival rate and adverse outcomes in CG patients. SLC6A14, a general amino acid transporter, can import all the essential amino acids in a manner dependent on the NaCl-generated osmotic gradients. Herein, we constructed GC cell sublines with high (SGC7901-M and MKN28-M) and low (MKN28-NM and SGC7901-NM) metastatic ability. Putative functional genes advancing GC metastasis were identified using mRNA microarray analysis and High-Content Screening. In particular, most significant change with a dampening trend in the migration potentiality of GC cells emerged after SLC6A14 gene was silenced. SLC6A14 expression was positively correlated with the migrated capability of different GC cell lines, and SLC6A14 was also constitutively expressed in GC patients with venous or lymphatic invasion, lymph node, or distant metastasis and poor prognosis, thus prompting SLC6A14 as a nonnegligible presence in supporting GC migration and invasion. Consistently, SLC6A14 depletion drastically depressed GC metastasis in vitro and in vivo. Most importantly, pharmacological blockade and gene silence of SLC6A14 both restricted epithelial-mesenchymal transition- (EMT-) driven GC metastasis, in which attenuated activation of the PI3K/AKT/mTORC1 pathway caused by amino acid starvation was involved. In summary, it is conceivable that targeting SLC6A14 has a tremendous promising for the treatment of metastatic GC.

In Cystic Fibrosis (CF) patients, inherited defects in anion transport leads to hyperactive epithelial Na+ channel (ENaC) activity. ENaC dysfunction in CF lung is known to cause airway surface liquid dehydration. Thick and dry mucus, in turn, leads to an inflammatory lung phenotype that often occurs without detection of infection in CF patients. Many investigative groups have reported that airway tissue and fluid from murine models exhibiting the CF lung phenotype (Scnn1b‐Tr mice), as well as CF patients, have increased levels of high mobility group box‐1 (HMGB‐1). HMGB‐1 is a cytokine mediator of inflammation and is a biomarker for severity of CF lung disorder. Herein, we hypothesize that HMGB‐1 plays an important role in the pathogenic regulation of ENaC, inflammation, and fibrosis in sterile airways. To begin testing this hypothesis, we measured transepithelial sodium current (Isc) and single channel ENaC open probability (Po) in human small airway epithelial cells (SAEC) in the presence or absence of 1 mg/mL human HMGB‐1 peptide. Electrophysiological measurements show that HMGB‐1 significantly increased amiloride‐sensitive ENaC Isc from −9.7±1.4 μA/cm2 to −18.4±2.9 μA/cm2 in confluent monolayers of SAEC (n=12; p=0.01) within 30 min. Subsequent amiloride treatment of the same SEAC monolayers decreased Isc 71%. Single channel measurements were conducted in primary SAEC obtained from 4 separate donor lungs in the cell‐attached configuration; HMGB‐1 increased ENaC Po from 0.18±0.02 to 0.35±0.04 (n=17; p&lt;0.01) in single channel analysis. Mean absolute total cell counts were significantly higher in bronchoalveolar lavage fluid (BALF) from mice intraperitoneally (IP) injected with HMBG‐1 (10 mg in 1 mL saline/kg) vs vehicle injected mice. Total immune cell counts were 27.4×103± 8.8×103 (in HMGB‐1) vs. 5.2×103±1.3X103 (in vehicle) injected mice (n=8 counts from 2 independent studies; p&lt;0.05). Masson's Trichrome labeling of IP injected mice show that HMGB‐1 significantly increases pulmonary fibrosis (see Fig 1). Flow cytometric analysis of HMGB‐1 and vehicle injected mice showed significant increase in IL‐1β, IL‐10, IL‐6, IL‐27, IL‐17A, IFN‐β, and GM‐CSF (n=3; p&lt;0.05; see Fig 2). Together, our data suggests that HMGB‐1 plays an important role in the CF lung phenotype; additional studies are underway to strengthen these observations. Impact: Successful completion of our work can lead to novel therapies needed to counteract ENaC dysfunction and inflammation in the CF lung.Support or Funding InformationThis work was supported by NIH 1 R01 HL137033‐01A1 awarded to MH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In May 2018, a seismically quiet region of the Indian Ocean awoke. More than 130 magnitude 4+ earthquakes were recorded in the first month, including a MW 5.9 event on May 15th, 2018. This seismic activity was later identified as being related to an exceptional underwater volcanic eruption offshore Mayotte island, which had emitted more than 6.5 km3 of lava by the time of writing. To better constrain the geodynamic processes responsible for the seismic and volcanic activity, a new network of ocean-bottom seismometers and land stations has been deployed around the seismically active region since February 2019. We present here an improved 1D velocity model for the active area and relocations of manually-picked earthquakes using this new model. The best-constrained events image detailed structures within two clusters of seismic activity east of Mayotte. The westernmost, proximal cluster, close to Mayotte's Petite-Terre island, has a “donut” shape horizontally and an “hourglass” shape in depth. The events distribution suggests the presence of a magma reservoir at around 27 km depth, with earthquakes focused along its sides, and a collapsing system underneath, related to the drainage of another, deeper magma storage zone. The distal cluster, focused 30–50 km offshore of Petite-Terre island, highlights the propagation of a dike between 45 and 25 km depth, aligned towards the new volcanic activity on the seafloor. We interpret this cluster as the fluid pathway towards the new volcano and nearby active seafloor lava fields. The improved velocity model also permits more robust daily monitoring of the seismicity using land stations, allowing local authorities to better assess seismic and volcanic hazards and to communicate them to the island's population.

Traubenwelke (berry shrivel, grape wilting), a physiological disorder during berry ripening, is a severe threat to grape production in numerous winegrowing areas worldwide. Investigations in Austrian viticulture, focusing on the particularly sensitive grape cultivar 'Blauer Zweigelt' ( Vitis vinifera L.), showed that afflicted clusters exhibited insufficient colouring, loss of turgor, low sugar content and high acidity. The aim of this study was to determine the chronological disease development on the basis of the process of sugar accumulation in both unhealthy and healthy clusters in relation to the emergence of visual symptoms. Sugar accumulation in afflicted clusters stopped shortly after veraison, well (approx. 20 days) before visible symptoms were detectable, and subsequently the degree of ripeness remained unchanged until harvest. The results suggested that development of Traubenwelke initiated before veraison. Distal clusters were more frequently affected than proximal clusters, whereas the position of the shoot on the vine did not impact frequency of occurrence. The gas exchange of leaves positioned opposite to healthy clusters was not significantly different than that of leaves opposite to unhealthy clusters.

The seismic sequence off the coast of Mayotte island, in the Comoros archipelago, preceded and accompanied the large submarine volcanic eruption and birth of the volcano Fani Maor&amp;#233;. While this sequence has slowed down and the eruption has stopped, it is still active and captures the deep complex system of this new volcano and its evolution. The seismicity is separated in two clusters. The distal cluster is located about 25 km South-East of Mayotte and has been associated with the magma propagation towards the surface. The proximal cluster, about 10 km East of Mayotte, suggests the presence of several magmatic reservoirs and conduits.&amp;#160;We built a new catalog using deep learning methods from land and ocean bottom seismometers data from 2019 to 2023. We locate this seismicity using NonLinLoc and a 1D velocity model developed in 2020 specifically for this seismic sequence. We analyze the volcano-tectonic and long period earthquakes spatio-temporal patterns as well as their mechanism to understand the volcanic system. We compare this analysis with recent modeling studies that suggest interactions between reservoirs.

In this longitudinal study, the authors addressed intracultural variation on fathers' interactions with their 3-month-old infants, their ideas about parental care, and the timing of their children's self-recognition at the age of 18-20 months. Participants were 24 middle-class German fathers and their firstborn children. Two behavioral clusters emerged: a more proximal parenting style with extensive body contact and a more distal parenting style with extensive object stimulation. Fathers in the distal cluster had significantly more education than did fathers in the proximal cluster. Children who had experienced more distal parenting were more likely to recognize themselves in a mirror than were children with more proximal parenting. The authors discuss the results with respect to fathers' influence on child development and patterns of intracultural variation.