Department of Clinical Biology, Tohoku University Graduate School of Medicine, Miyagi, Japan; Division of Medical Science, Tohoku University Graduate School of Biomedical Engineering, Miyagi, Japan; andDepartment of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine, Miyagi, Japan.
Nephronophthisis (NPHP)-related ciliopathies are recessive, single-gene disorders that collectively make up the most common genetic cause of CKD in the first three decades of life. Mutations in 1 of the 15 known NPHP genes explain less than half of all cases with this phenotype, however, and the recently identified genetic causes are exceedingly rare. As a result, a strategy to identify single-gene causes of NPHP-related ciliopathies in single affected families is needed. Although whole-exome resequencing facilitates the identification of disease genes, the large number of detected genetic variants hampers its use. Here, we overcome this limitation by combining homozygosity mapping with whole-exome resequencing in a sibling pair with an NPHP-related ciliopathy. Whole-exome capture revealed a homozygous splice acceptor site mutation (c.698G>T) in the renal Mg2+ transporter SLC41A1. This mutation resulted in skipping of exon 6 of SLC41A1, resulting in an in-frame deletion of a transmembrane helix. Transfection of cells with wild-type or mutant SLC41A1 revealed that deletion of exon 6 completely blocks the Mg2+ transport function of SLC41A1. Furthermore, in normal human kidney tissue, endogenous SLC41A1 specifically localized to renal tubules situated at the corticomedullary boundary, consistent with the region of cystogenesis observed in NPHP and related ciliopathies. Last, morpholino-mediated knockdown of slc41a1 expression in zebrafish resulted in ventral body curvature, hydrocephalus, and cystic kidneys, similar to the effects of knocking down other NPHP genes. Taken together, these data suggest that defects in the maintenance of renal Mg2+ homeostasis may lead to tubular defects that result in a phenotype similar to NPHP.
Rare CKDs make up the majority of CKD cases treated within long-term dialysis and renal transplantation programs in the first three decades of life but are notoriously difficult to diagnose.1 Rare recessive mutations cause chronic diseases that often require hospitalization.2 However, half of their single-gene causes are still unknown (http://omim.org/statistics/entries). Because recessive single-gene mutations directly represent the disease cause, gene identification offers a powerful approach to revealing disease mechanisms. Furthermore, because recessive mutations predominantly convey loss of function, recessive single-gene defects can be transferred directly into animal models to study the related disease mechanisms and to screen for small molecules as possible treatment modalities.
The finding that some of the more recently identified genetic causes of NPHP-related disorders are exceedingly rare necessitates a strategy to identify novel single-gene causes of these conditions in single affected families.15 In this context, the new method of whole exome capture with consecutive massively parallel sequencing (here called whole exome resequencing [WER]) theoretically offers a powerful approach toward gene identification in rare recessive diseases. However, the utility of WER is hampered by the large number of novel genetic variants that result from whole exome sequencing in any given individual.18,21 To overcome this limitation of WER, we developed a strategy that combines WER with homozygosity mapping.18 Using this approach, we have identified mutation of the renal magnesium transporter gene SLC41A1 as a novel genetic cause of disease that phenocopies NPHP-related conditions clinically, ultrasonographically, and histologically.
To identify additional causative mutations for NPHP-related disorders, we performed WER in a consanguineous Italian family (F438) with an apparent NPHP-related disorder phenotype (Figure 1A). In this large family, two siblings born from first cousins (IV1 and IV2) displayed the unusual association of apparent NPHP with bronchiectasis.
Renal ultrasonography and biopsy indicate characteristic hallmarks of nephronophthisis. (A) Pedigree for family F438. Individuals IV1 and IV2 display both NPHP and primary ciliary dyskinesia (shaded shapes). Individual IV3 has only primary ciliary dyskinesia (half-shaded). (B) RUS showing increased echogenicity, corticomedullary cysts, and loss of corticomedullary differentiation in patients F438-IV1 (left panel) and F438-IV2 (right panel). (C) Renal histologic findings from a renal biopsy specimen from F438-IV1 showing the characteristic triad of tubular ectasia, tubular basement membrane disruption, and tubulointerstitial infiltrations (Trichrome-Masson staining).
Patient IV2 had had frequent episodes of fever, coughing, and respiratory infections since the first month of life, and chest radiography demonstrated the persistence of inflammatory infiltrates in the lung. High-resolution computerized tomography revealed bronchiectasis, and fiberoptic bronchoscopy showed reddening of the bronchial mucosa with purulent secretion. Evaluation of bronchoalveolar lavage showed a marked increase in the percentage of neutrophils and the presence of Pseudomonas aeruginosa. At age 8 years, the patient was referred to the Nephrology Department because of observed polyuria and polydipsia. Initial urine analysis revealed low urine osmolarity (380 mOsmol/kg in fresh morning urine), and blood analysis displayed renal insufficiency with serum creatinine levels of 2.37 mg/dl. Renal ultrasonography showed bilateral irregular echogenicity, with both kidneys decreased in size (Figure 1B). Percutaneous renal biopsy showed periglomerular fibrosis, tubular ectasia, tubular basement membrane disruption, and tubulointerstitial infiltrations (Figure 1C). On the basis of these clinical findings, a diagnosis of NPHP was proposed. The patient underwent peritoneal dialysis at age 9 years before undergoing kidney transplantation at age 10.
Patient IV1, like his sibling, had had a history of fever, coughing, and respiratory infections since the first month of life. IV1 also presented with polyuria and polydipsia with low urine osmolarity (420 mOsmol/kg) and serum creatinine level of 1.5 mg/dl at 5 years of age. Renal biopsy and renal ultrasonography showed similar findings as for patient IV2 (Figure 1B). Progression of renal damage led to ESRD at age 10 and initiation of peritoneal dialysis.
Patient IV3 is a first-line cousin of siblings IV1 and IV2. She presented with chronic bronchitis and bronchiectasis at age 6 years. However, by age 11 no signs of polyuria or polydipsia were present and results of blood and urine analysis were normal. In addition, renal ultrasonography revealed no abnormalities in kidney size or structure.
Homozygosity mapping performed on patient IV1 yielded eight homozygosity peaks (Figure 2A). By WER, we detected four homozygous variants (Supplemental Table 1) whose segregation pattern fit a recessive mode of inheritance. Of these four variants, only the variant in the SLC41A1 gene was not a known single-nucleotide polymorphism (SNP) and was absent from both the 1000 Genomes and Exome Variant Server databases (Supplemental Table 2). Furthermore, only the SLC41A1 variant was predicted to be damaging by all four predictive tools used (Polyphen 1, Polyphen 2, MutationTaster, SIFT [Supplemental Table 2]). In addition, only two of the variants (SLC41A1 and ZNF224) display strong evolutionary conservation at the mutated residue (Supplemental Table 3). Finally, the homozygous missense mutation (c.698G>T, p.G223V) in SLC41A1 resides within an evolutionary conserved exonic splice acceptor site in exon 6 of SLC41A1, probably resulting in missplicing of the SLC41A1 transcript and thus a potentially more deleterious mutation. This mutation was absent from 100 European-American healthy controls and also absent from 80 regionally matched healthy controls (G. Caridi, personal communication). These data together suggest that SLC41A1 as a novel causative gene of an NPHP-related disorder phenotype (Figure 2, B and C).
Recessive mutation in SLC41A1 in family F438 identified by whole exome capture and homozygosity mapping. (A) Homozygosity profile for patient IV1. Nonparametric logarithm of odds scores were plotted over genetic distance across the genome, where chromosomal positions are concatenated from p- to q-arm (left to right). Arrowhead shows the SLC41A1 locus. (B) Diagram of the human SLC41A1 gene showing exon number and primer locations. Red arrow shows position of the c.698G>T splice acceptor site mutation. (C) Sanger-sequencing confirmation of the c.698G>T mutation in SLC41A1. Altered nucleotide and amino acid change are given above the sequence trace. WT control sequence and trace are shown below the mutated sequence. Codon triplets are underlined to indicate reading frame. Noncoding sequence is in lower case. Mutated nucleotide is denoted by arrowhead. (D) RT-PCR was performed on RNA from lymphoblastoid cells prepared from parents (III5 and III6) and affected siblings (IV1 and IV2) using the primers indicated. Positions of primers is indicated in B. M, 100-bp DNA ladder. (E) Sanger sequencing of the fast and slow migrating RT-PCR products (primers P1 + P3) revealed that the c.698G>T mutation results in skipping of exon 6 (upper trace).
To test whether the c.698G>T mutation impairs normal splicing of the SLC41A1 transcript, Epstein Barr virus (EBV)–transformed white blood cells were prepared from blood samples from both parents (III5 and III6) and the two affected siblings (IV1 and IV2). RT-PCR was performed using primers flanking exon 6 (Figure 2B) on cDNA prepared from both the EBV-transformed white blood cells. This test revealed that in both parents, one PCR product of the expected size and an additional faster-migrating PCR product was observed (Figure 2D, upper panel), consistent with the fact that both parents are heterozygous for the c.698G>T mutation. In contrast, RT-PCR on patient-derived cDNA yielded only the single faster-migrating band (Figure 2D, upper panel). Sanger sequencing of this faster-migrating band revealed that it arises from skipping of SLC41A1 exon6 (Figure 2E). Consistent with this observation, further RT-PCR using a forward primer designed against exon 6 (P2; Figure 2B), revealed no detectable PCR product in the two affected siblings but did show a PCR product of the expected size in the two parents (Figure 2D, middle panel). Together, these data demonstrate that the c.698G>T mutation results in missplicing of the SLC41A1 transcript.
The SLC41A1 mutations that results in skipping of exon 6 is predicted to result in an in-frame deletion of a transmembrane domain. We next tested whether this deletion abrogates expression of SLC41A1. Western blotting of protein lysates generated from both parental and patient-derived cell lines revealed almost complete loss of SLC41A1 expression in the two affected siblings (Figure 3A).
The c.698G>T mutation results in reduced expression and loss of function of SLC41A1. (A) Western blot of lysates prepared from parents (III5 and III6) and affected siblings (IV1 and IV2) showing decreased expression of SLC41A1 in affected siblings. Anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Western blot was used as loading control. (B) EGFP-tagged WT SLC41A1 and SLC41A1 lacking exon 6 (dEx6) were transiently transfected into MDCKII cells. Cells were fixed and EGFP fluorescence captured by confocal microscopy. (C) HEK293 cells transiently transfected with WT or dEx6 FLAG-tagged SLC41A1 were biotinylated at the cell surface before lysis. Cell lysates were then subjected to pulldown with neutravidin beads before SDS-PAGE and Western blotting with the indicated antibodies. (D) HEK293 cells were transfected with empty vector (MOCK) WT or dEx6 EGFP-SLC41A1 before loading with mag-fura2-AM. To measure magnesium influx, cells were exposed to high Mg2+, low Na+ solution, and radiometric fluorescent images were collected to determine intracellular [Mg2+]. (E) To measure magnesium efflux, cells were treated as above and then switched into magnesium-free media, and radiometric fluorescent images were collected to determine intracellular [Mg2+]. Error bars show standard deviation.
We next tested whether the mutated form of SLC41A1 can traffic in a similar fashion as the WT protein. An amino-terminal enhanced green fluorescent protein (EGFP)–tagged SLC41A1 expression construct was generated by cloning the full-length (WT) human SLC41A1 open-reading frame into a mammalian expression vector. In addition, an expression construct with the residues encoded by SLC41A1 exon 6 deleted was cloned from patient-derived cDNA (dEx6). Transfection of the WT and mutant constructs into the MDCKII renal epithelial cell line revealed that both targeted to the basolateral membrane and that the mutant localization was indistinguishable from WT (Figure 3B). Furthermore, using a cell-surface biotinylation assay in which HEK293 cells were transfected with WT or mutant (dEx6) FLAG-tagged SLC41A1, neutravidin pulldown revealed that both the WT and mutant SLC41A1 are trafficked to the cell surface (Figure 3C). These data suggest that the in-frame deletion of exon 6 does not affect the normal trafficking of SLC41A1.
Because the in-frame deletion of exon6 did not appear to perturb the normal trafficking of SLC41A1, we next sought to address whether the magnesium-transporting ability of SLC41A1 might be compromised by this deletion. In cells transfected with WT SLC41A1, basal free intracellular Mg2+ ([Mg2+]i) was, on average, 1.12±0.06 mM (n=20), which was significantly higher than that of mock transfected cells (0.81±0.02 mM, n=20). The average basal [Mg2+]i of mutant transfected (ΔE6) cells, 0.87±0.04 (n=33), was very similar to that of mock cells (Figure 3D). To examine the Mg2+ transport activity, cells were exposed to high Mg2+, low Na+ solution (Mg2+ influx). A 15-minute exposure to high Mg2+, low Na+ solution resulted in a robust elevation of [Mg2+]i in WT cells, whereas only small increases in [Mg2+]i were observed in mock and ΔE6 cells (WT, 0.50±0.03 μM/s; mock, 0.21±0.04 μM/s; ΔE6, 0.20±0.01 μM/s). When the extracellular solution was switched to the Ca2+-free Tyrode solution, [Mg2+]i returned to the basal level in WT, ΔE6, and mock cells (Mg2+ efflux). Again, Mg2+ efflux rates were similar in mock cells and ΔE6 cells (Figure 3E). (Comparison of the efflux rates between WT cells and others is difficult because Mg2+ efflux was induced at much higher initial [Mg2+]i in WT cells.) These data indicate that WT has Mg2+ transport activities, whereas ΔE6 mutant has null function.
One of the hallmarks or NPHP is the formation of renal cortico-medullary cysts.3,22 To ascertain why loss of SLC41A1 activity may cause the appearance of cysts at the cortico-medullary boundary, we examined whether SLC41A1 is expressed in nephron segments in this region. Indeed, immunohistochemical analysis of SLC41A1 expression on normal human kidney sections revealed expression primarily in the distal convoluted tubules (DCTs) and in the tubule adjacent to the macula densa (Figure 4A). Of note, claudin16, a major regulator of renal paracellular magnesium uptake in which recessive mutations cause renal disease that closely mimics NPHP in cattle,23 was coexpressed in identical tubular segments to SLC41A1 in rat kidney sections (Figure 4B). In previous studies, RT-PCR has shown that claudin16 is expressed primarily in the DCTs and thick ascending limb of nephrons,22 and indeed RT-PCR confirmed that SLC41A1 is also expressed in the DCT and thick ascending limb but is absent from proximal convoluted tubule and cortical collecting duct of microdissected tubular segments (Figure 4C). Together, these data reveal that SLC41A1 is expressed in the DCT, macula densa, and thick ascending limb tubular segments, whose location is entirely consistent with the region of cystogenesis observed in NPHP and related ciliopathies.
SLC41A1 localizes predominantly to distal convoluted tubules. (A) Immunohistochemistry was performed on human paraffin-embedded sections using rabbit anti-SLC41A1 antibodies. Signal was seen primarily in the DCTs. G, glomerulus. Arrowhead shows macula densa. (B) Rat paraffin-embedded sections were immunostained with antibodies against SLC41A1 (red) and Claudin 16 (green). DAPI (blue) was used to stain cell nuclei. (C) RT-PCR was performed on human nephron segments with primers for SLC41A1 and actin-β (ACTB) as loading control. CCD, cortical collecting duct; NTC, no template control; PCT, proximal convoluted tubule; TAL, thick ascending limb.
To confirm that defective SLC41A1 function can lead to cystogenesis, we examined SLC41A1 function in the zebrafish, a widely used model organism for the study of human ciliopathies. Alignment of the human SLC41A1 protein with the zebrafish homolog revealed extensive conservation with 76% identity at the protein level, suggesting that the zebrafish homolog is likely to function in a manner similar to that of human protein (Supplemental Figure 1). To examine the expression pattern of slc41a1 in zebrafish, an antisense in situ probe was generated. By in situ hybridization, slc41a1 was expressed almost exclusively in the corpuscle of Stannius of the zebrafish pronephros, as previously described24 (Figure 5A). To ablate slc41a1 expression, a splice-blocking morpholino designed against the splice acceptor site of exon 4 (Figure 5B) was injected into 1–2 cell stage zebrafish embryos. Twenty-four hours after injection, embryos were harvested and the efficacy of the morpholino was examined by RT-PCR. RT-PCR of RNA prepared from embryos injected with a standard control morpholino revealed a PCR product of the expected size, whereas embryos injected with the splice-blocking morpholino exhibited a faster-migrating product indicative of missplicing consistent with skipping of exon 4 (Figure 5C).
Knockdown of slc41a1 expression in zebrafish results in kidney cysts. (A) In situ hybridization of 48-hour-old zebrafish embryos with an antisense slc41a1 probe showing expression in the corpuscle of Stannius (arrowhead). (B) Schematic diagram of the zebrafish slc41a1 gene showing location of the splice-blocking morpholino (MO) and primers used for RT-PCR (P1 and P2). Shown below are expected RT-PCR products using primers P1 and P2 on WT and splice morpholino injected (dEx4) samples and their expected sizes. (C) RT-PCR was performed on RNA prepared from embryos 24 hours after injection with standard control (Con) or slc41a1 splice-blocking morpholino (SLC). Shown on the left are the sizes (in bp) of the major bands of the DNA ladder (M). NTC, no template control. (D) Phenotypic appearance at 48 hours of control and slc41a1 morpholino–injected embryos. (E) Methylene blue–stained plastic sections showing prominent hydrocephalus in slc41a1 morpholino–injected embryos (right panel) compared with control morpholino–injected embryos (left panel). (F) Methylene blue–stained plastic sections showing tubular dilation (cysts) in slc41a1 morpholino–injected embryos (right panel) compared with control morpholino–injected embryos (left panel). Arrowheads show pronephric tubules.
To examine the effect of loss of slc41a1 expression, 48 hours after morpholino injection, the morphology of control and slc41a1 morphants was examined. Control morphants examined no obvious phenotype, whereas slc41a1 morphants displayed severe developmental abnormalities (Figure 5, D and E). Specifically, >70% of slc41a1 morphants exhibited severe ventral body curvature, a phenotype frequently observed upon knockdown of NPHP genes, compared with <2% of embryos injected with the control morpholino (Figure 5D and Supplemental Table 4). In addition, a high proportion (>50%) of the slc41a1 morphants displayed severe hydrocephalus compared with control morphants (Figure 5D). Morphologic analysis of sections through the hindbrain of slc41a1 morphants showed extensive dilation of the ventricle compared with controls (Figure 5E).
NPHP-related disorders (including Bardet Biedl, Joubert, Senior-Loken, and Meckel syndromes) display significant locus heterogeneity, with causative mutations occurring in more than 30 different genes. Causative mutation frequency among these genes is not homogenous, ranging from frequent mutations (NPHP1, mutated in about 20% of all cases of NPHP-related disorders in our cohort) to extremely rare mutations (GLIS2/NPHP7, mutated in a single family).15,26 It would appear that mutation of SLC41A1 represents an extremely rare cause of NPHP-related disorders similar to GLIS2/NPHP7, as screening of 1000 patients with NPHP-related disorders using a newly developed high-throughput sequencing technique failed to identify any additional patients with SLC41A1 mutations.27 Furthermore, it is likely that the identified splice site mutation in SLC41A1, resulting in the skipping of exon 6, may be hypomorphic because a small amount of protein product was still detectable in the two affected patients (Figure 3A). Consequently, one may postulate that null mutations resulting from nonsense mutations or deletions might be embryonic lethal and hence not be represented in our cohort of patients with NPHP-RC.
What remains to be ascertained in this case is the cause of the bronchiectasis in the two affected siblings and their cousin. The identified mutation in SLC41A1 can explain the renal phenotype of the two affected siblings but cannot explain the cause of bronchiectasis because the cousin who had lung dysfunction but no renal disease is heterozygous for the SLC41A1 mutation. A dominant effect for the SLC41A1 allele is unlikely because neither parent of the two affected siblings who are heterozygous for the mutant SLC41A1 allele showed any bronchiectasis phenotype. The possibility that the cousin had a compound heterozygous mutation in SLC41A1 was ruled out as Sanger sequencing of all exons failed to identify additional mutations (data not show). The bronchiectasis phenotype probably arises from mutation of a separate gene. Using our exome data, we searched for mutations in all known primary ciliary dyskinesia genes, and although we did find variations in several, none of them segregated correctly with affected status within the family (Supplemental Table 5). Alternatively, the respiratory problems observed in the cousin (IV3) may be a phenocopy of the disease observed in the two siblings (IV1 and IV2). This is supported by the observation that the two affected siblings displayed symptoms of lung dysfunction soon after birth, whereas the cousin only exhibited problems at age 6 years.
Intracellular Mg2+ is a cofactor for enzymes and signal transduction proteins and regulates bioenergetics, ion transport, growth, and proliferation. [Mg2+]i is thought to be maintained by transport across cell membranes with Mg2+ transporters. SLC41A1, a member of the solute carrier family 41, is a cell membrane protein expressed in various tissues, including heart, brain, kidney, liver, and colon, and has been proposed as one of the candidates for such Mg2+ transporters.28–30 Although Mg2+ transport by SLC41A1 has not been fully characterized, Kolisek et al. recently reported that the human SLC41A1 gene encodes for the Na+/Mg2+ exchanger 4.28 The finding that mutations of this renal Mg2+ transporter mimics an NPHP-RC phenotype may be related to the fact that other mutations in proteins involved in renal Mg2+ transport are known to cause an NPHP-related disorder phenotype. Specifically, recessive mutations of the CLDN16 gene encoding the renal tight junction protein claudin-16 (also called paracellin-1) cause a recessive renal disease that closely mimics NPHP31 in cattle24 both clinically and histologically. An unusual finding, given that SLC41A1 is an apparent magnesium transporter, is that neither of the affected patients exhibited any abnormalities in serum or urine magnesium content or any evidence of nephrocalcinosis (G. Caridi, personal communication). Hence, the disease phenotype may result from perturbed intracellular magnesium homeostasis rather than global alterations in magnesium levels.
It is unclear how loss of correct magnesium homeostasis within the epithelial cells of the distal convoluted tubule could lead to the formation of cysts and the development of the NPHP-related disorder phenotype. Because the majority of cases of NPHP-related disorders are caused by mutations in genes whose products reside at or regulate ciliary function, one possibility is that the SLC41A1 transporter is regulated downstream of the NPHP-protein/ciliary network. Analysis of whether cellular magnesium levels are perturbed upon loss of cilia or knockdown of other NPHP proteins could potentially provide important insight into the pathogenesis of NPHP-related disorders. Of note, SLC41A1 is the first transporter to be identified as causing an NPHP-related disorder, and as such it represents an attractive potential target for pharmacologic intervention for the treatment of these disorders.
For genome-wide homozygosity mapping35 the Genome-wide Human SNP 6.0 ArrayII from Affymetrix was used. Genomic DNA samples were hybridized and scanned using the manufacturer's standard protocol at the University of Michigan Core Facility (www.michiganmicroarray.com). Nonparametric logarithm-of-odds scores were calculated using a modified version of the program GENEHUNTER 2.136,37 through stepwise use of a sliding window with sets of 110 SNPs using the program ALLEGRO.38 Genetic regions of homozygosity by descent (homozygosity peaks) were plotted across the genome as candidate regions for recessive disease-causing genes. Disease allele frequency was set at 0.0001, and Caucasian marker allele frequencies were used.
Exome enrichment was conducted following the manufacturer's protocol for the NimbleGen SeqCap EZ Exome v2 beads (Roche NimbleGen Inc.). The kit interrogates a total of approximately 30,000 genes (about 330,000 consensus coding DNA sequence exons). Massively parallel sequencing was performed largely as described in Bentley et al.39 Sequence reads were mapped to the human reference genome assembly (National Center for Biotechnology Information build 36/hg18) and mutation calling performed using CLC Genomics Workbench (version 4.7.2) software (CLC bio, Aarhus, Denmark).
HEK293T and MDCK II cells were cultured in DMEM with 10% FBS in 5% CO2 at 37°C. Transfection was performed using Lipofectamine 2000 (Invitrogen). Cell surface biotinylation assay was performed with the Cell Surface Protein Isolation kit (Thermo Scientific) according to the manufacturer's protocol.
Human SLC41A1 was cloned from human kidney cDNA (Clontech). Exon 6–deleted SLC41A1 was cloned from the cDNA made from EBV-transformed proband's peripheral lymphocytes. SLC41A1 cDNAs were subcloned into pEGFP-C (Invitrogen) or FLAG-tagged pcDNA3.1.
Transfected cells were grown on 35-mm glass base dishes (Iwaki, Chiba, Japan) coated with Matrigel (BD Biosciences). Cells were washed with Ca2+-free Tyrode solution composed of (in mM): 135 NaCl, 5.4 KCl, 1.0 MgCl2, 0.33 NaH2PO4, 0.1 EGTA, 10 HEPES, and 2 glucose (pH 7.4 at 25°C by NaOH). After the background fluorescence of the GFP-positive transfected cells were measured, cells were incubated with 5 μM mag-fura-2-AM for 10 minutes, followed by three washes and further 20-minute incubation in the Ca2+-free Tyrode solution on the stage of inverted microscope (TE300; Nikon, Tokyo, Japan). The fluorescence images of mag-fura-2 were collected from the cells at 25°C by a cooled charge-coupled device system (EM-CCD C9100, Hamamatsu Photonics, Hamamatsu, Japan) with a 40× objective (S fluor 40×0.90, Nikon). With alternate excitation at 345 and 380 nm, mag-fura-2 fluorescence images at wavelength longer than 470 nm were acquired, and digitized data were analyzed with image analysis software (Aquacosmos/Ratio, Hamamatsu Photonics). The ratio of fluorescence intensities excited at 380 nm and 345 nm was calibrated in terms of [Mg2+]i as described previously.40 To assess Mg2+ influx, cells were exposed to a high Mg2+, low Na+ solution composed of (in mM): 68.5 MgCl2, 24 Mg-methanesulfonate, 5.4 KCl, 0.33 NaH2PO4, 0.1 EGTA, 10 HEPES, and 2 glucose (pH 7.4), and fluorescence images were obtained at 0, 5, 10, and 15 minutes. To assess Mg2+ efflux, extracellular solutions of the Mg2+-loaded cells were changed back to the Ca2+-free Tyrode solution and fluorescence images were obtained every 10 seconds for 20 minutes.
To knock down zebrafish slc41a1, a splice-site blocking morpholino was designed against the slc41a1 exon 4 splice acceptor site (TGAACCTGAACACAGCAGAGGGACA). Morpholinos were dissolved in nuclease-free water and injected into zebrafish embryos at 1- to 4-cell stages in 0.1 M KCl at the specified dosage. The injection volume is estimated to be 1–2 nl.
Kidney cortex tissues were obtained during unilateral nephrectomy for renal carcinoma.41 The institutional review board of University of Tokyo School of Medicine approved the study. Human nephron segments were obtained from manual microdissection, as described previously.42 Total RNA isolated from each nephron segments was used for RT-PCR. Primers were 5′-ggtcttcatcctagtgcctg and 5′-caaggtgatgaggtcgcc for SLC41A1; 5′-cgcaaagacctgtacgccaac and 5′-ccacacggagtacttgcgctc for ACTB.
Zebrafish embryos were fixed with 4% paraformaldehyde overnight, serial-dehydrated with 25%, 50%, 75%, and 95% ethanol, and then equilibrated with JB-4 solution (Polysciences) overnight at 4°C. The embryos were embedded in JB-4 resin and sectioned with a Leica R2265 microtome. The sections were stained with methylene blue as previously described.25 For immunohistochemistry of human and rat kidney heat-induced antigen retrieval was done, and then paraffin-embedded human and rat kidney sections were stained with anti-SLC41A1 (Sigma Aldrich, St. Louis, MO) and Claudin 16 (Novus Biologicals, Littleton, CO). EnVision+ Dual Link System peroxidase (DAKO, Carpinteria, CA) and 3,3′-diaminobenzidine were used to visualize staining in human tissue. For rat kidney sections, goat antimouse A488 and goat antirabbit A594 (Invitrogen) secondary antibodies were used.
The authors thank the families who contributed to this study. We thank Robert H. Lyons for excellent Sanger sequencing.
This research was supported by grants from the National Institutes of Health to F.H. (DK1069274, DK1068306, DK064614), W.Z. (DK091405), and E.A.O. (DK090917), and by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to T.A. (23390033). F.H. is an investigator of the Howard Hughes Medical Institute, a Doris Duke Distinguished Clinical Scientist, and a Frederick G.L. Huetwell Professor.
T.W.H. and E.A.O. contributed equally to this work.
See related editorial, "Working Out Nephronophthisis Genetics One Family at a Time," on pages 865–868.
This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2012101034/-/DCSupplemental.
Carrier testing for severe childhood recessive diseases by next-generation sequencing. Sci Transl Med 3: 65ra4, 2011.


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The Atlanta Symphony Orchestra will perform as part of the Summer MusicFest series on the lawn of the Smithgall Arts Center in Gainesville on Friday, June 20, at 8 p.m. Tickets are $35 for adults; $32 for seniors; and $28 for students. Visit http://www.theartscouncil.net/aso.html or call 770-534-2787 to purchase tickets and learn more information.
Gates open at 6 p.m. to those who would like to picnic on the lawn before the show. Bring a picnic dinner or purchase one from local downtown restaurants. In the event of inclement weather, the concert will move indoors to Pearce Auditorium.


The New Year's Day walk is a classic for a reason. It's your chance to clear your mind and reset for the year ahead. It sets you up to achieve anything you want in the new year. It's also the perfect way to make sure you're ticking off your resolutions straight away, making memories with your nearest and dearest from day one of the new year.
This New Year's Day, join us for First Foot.
In Northern British folklore, the first-foot is a coveted prize. As the clock strikes twelve and brings in a new year, the first person to pass the threshold of your home claims the title of your first-foot, bringing you luck for the year ahead. However, this tradition runs the risk of being lost for good, and we're not letting that happen.
For 2018, help us to turn the first-foot tradition upside down. Instead of focusing on the first person that comes inside your home, why don't we make our first-foot the person we first step outdoors with?
This New Year's Day, we want you to get out there on a hike with your family or friends (furry ones count too!), and put your First Foot into the outdoors. That's right. Grab your mum, your dad, your uncle, your aunt and get out there. And let's start the new year with something a little more strenuous than the usual New Year's Day walk. You only get one First Foot a year, so make it count and tick off hike number one for 2018.
Together, we'll make sure our fresh take on the First Foot tradition lives on.


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Fund Alert, December 18, 2017
SEC Delays Filing Requirement for Form N-PORT
On Dec. 8, 2017, the Securities and Exchange Commission (the Commission) issued a release delaying EDGAR filing requirements for Form N-PORT for nine months as a result of cybersecurity concerns.1 Consequently, larger fund groups (those with net assets of at least $1 billion) will be required to begin submitting reports on Form N-PORT via EDGAR by April 30, 2019 (as opposed to the original requirement to file by July 30, 2018), and smaller fund groups will be required to begin submitting reports on Form N-PORT by April 30, 2020 (originally July 30, 2019). During the delay, funds will continue to file Form N-Q. The June 1, 2018, compliance date for filing Form N-CEN was not delayed.
During the nine-month delay, larger fund groups technically will continue to be subject to the Form N-PORT requirement, and their compliance date will not change. In lieu of filing reports with the Commission, Temporary Rule 30b1-9(T) will require larger fund groups to maintain in their records the information that is required to be included in Form N-PORT. While this information is not required to be stored in the extensible markup language, or XML, format required by Form N-PORT, larger fund groups must still prepare and maintain in their records the information that is required to be included on Form N-PORT.2 However, the Commission suggests that maintaining the information in an XML format would facilitate the filing of Form N-PORT following the nine-month delay, as funds can use the delay to gain greater facility with the form's structured reporting format.3 The maintained information will be subject to Commission recordkeeping requirements4 and, like all such records, will be subject to examination by Commission staff.5 Accordingly, it is important that the information required to be compiled for Form N-PORT during this delay be maintained in an easily accessible and, presumably, centralized location.
Smaller fund groups will receive an extension of their compliance date and will not be subject to a requirement to maintain the information required by Form N-PORT during the delay.
New Compliance and Filing Due Dates
Larger Fund Groups Smaller Fund Groups
N-PORT – compliance date June 1, 2018 (unchanged) March 1, 2020
N-PORT – initial filing due date (reporting period) April 30, 2019
(month ending March 31, 2019) April 30, 2020
(month ending March 31, 2020)
N-PORT – initial filing with information made publicly available (reporting period) Oct. 30, 2019
(month ending Sept. 30, 2019)6 April 30, 2020
N-CEN – initial filing due date 75 days after the end of the fiscal year ending on or after June 1, 2018 (unchanged) 75 days after the end of the fiscal year ending on or after June 1, 2018 (unchanged)
Background and Reasons for the Delay
On Oct. 13, 2016, the Commission adopted new rules and forms, including Form N-PORT, that were designed to modernize the reporting and disclosure of information by investment companies.7 Form N-PORT requires reporting of a fund's complete portfolio holdings and additional information that will facilitate risk analysis and other Commission oversight. Reports on Form N-PORT are required to be filed in an XML-structured data format no later than 30 days after the close of each month, using the Commission's EDGAR system.8 In general, reports on Form N-PORT for every third month of each fiscal quarter will be available to the public 60 days after the end of the fiscal quarter, but reports for the first and second months of each quarter and portions of reports for the third month will be kept nonpublic permanently.9
The Commission stated in the Release announcing the delay that the delay takes into account sound data security practices and protocols for sensitive, nonpublic information.10 Notably, this delay follows a recent disclosure by Commission Chairman Jay Clayton that the EDGAR system was compromised by a cyberattack.11 The Commission is aware that some Form N-PORT data items could be competitively sensitive and that a breach could result in harm to the reporting funds.12 As the Commission works to adopt measures designed to improve EDGAR's functionality and security, the Commission is concerned that these measures could negatively affect EDGAR's ability to validate and accept Form N-PORT filings in a timely manner, in particular during peak filing periods.13 Efforts to address any such potential effects are underway, according to the Commission, but the delay of Form N-PORT filing requirements provides time for the Commission to complete its review and to implement and test any resulting modifications to the EDGAR system.14
Details of the Delay
As a result of the Commission's action, funds in larger fund groups, which have a compliance date of June 1, 2018, and previously would have been required to submit their first reports on Form N-PORT on EDGAR for the month ending June 30, 2018 (no later than July 30, 2018), will not become subject to the requirement to file Form N-PORT on EDGAR until April 1, 2019. Thus, their first EDGAR filing will be for the month ending March 31, 2019, and will be due no later than April 30, 2019. Until that time, they will comply with their Form N-PORT obligation by maintaining the required information in their records under Temporary Rule 30b1-9(T). Funds in smaller fund groups will have their compliance date extended from June 1, 2019, to March 1, 2020, so they will not be subject to a Form N-PORT requirement until the month ending March 31, 2020 (due no later than April 30, 2020). Funds will continue to file public reports on Form N-Q until they begin filing reports on Form N-PORT using EDGAR.
When it adopted Form N-PORT, the Commission determined that having a six-month time period during which larger fund groups are required to file reports on Form N-PORT, but during which those reports are not disclosed publicly, will allow funds and the Commission to make adjustments to fine-tune the technical specifications and data validation processes.15 The Commission is preserving this six-month time period, so larger fund groups' reports for the periods ending March 31, 2019, through Sept. 30, 2019, will be kept nonpublic.16
The Commission did not modify the compliance or filing dates with respect to Form N-CEN, which will have its reports made public immediately upon filing and, therefore, does not raise the same cybersecurity concerns.17 The compliance date for Form N-CEN continues to be June 1, 2018.
Effect on Liquidity Risk Management Rule
The EDGAR reporting requirements added to Form N-PORT by the Commission when it adopted its liquidity risk management rule were also delayed by the Temporary Rule.18 That release adopted additional reporting requirements on Form N-PORT, specifically requiring a fund to report monthly position-level and aggregate liquidity classification information and its highly liquid investment minimum to the Commission. The liquidity rule and related reporting requirements have a compliance date of Dec. 1, 2018, for larger fund groups, and June 1, 2019, for smaller fund groups. In effect, therefore, both larger fund groups and smaller fund groups will have nine-month delays of the periods before which they must file liquidity information on Form N-PORT that is made publicly available.19 Larger fund families will only be required to comply with Temporary Rule 30b1-9(T) with respect to these liquidity-related additions to Form N-PORT based on the Dec. 1, 2018, compliance date.20
Several trade groups have submitted letters to the Commission requesting delay of significant parts of the liquidity risk management rule and its related reporting requirements.21 The delay for filing Form N-PORT will not preclude the Commission from considering the requests for delay contained in those letters.
1 Investment Company Reporting Modernization, Release Nos. 33-10442, 34-82241, IC-32936 (Dec. 8, 2017), 82 Fed. Reg. 58,731 (Dec. 14, 2017), ("Release").
2 Id. at 58,736.
3 Id. at 58,733 n.28.
4 Specifically, the information will be subject to Rule 31a-2(a)(2), which provides that funds must preserve applicable records for a period not less than six years from the end of the fiscal year, the first two years in an easily accessible place. As fund records, the information will be subject to the antifraud provisions of Section 34(b) of the Investment Company Act of 1940.
5 See Release, supra note 1, at 58,733.
6 Only reports for the third month of each fiscal quarter will have information made public. If the month ending Sept. 30, 2019, is not the third month of a fiscal quarter, then a later report will be first to have information made public.
In addition, we assume that the Commission did not intend the six-month period of nonpublic filings to include filings for the month ending Sept. 30, 2019. See infra note 16. If this is incorrect, then the first filing that may have information made public would be the filing for the month ending Oct. 31, 2019 (or the next filing that is for the third month of a fiscal quarter).
7 Investment Company Reporting Modernization, Release Nos. 33-10231, 34-79095, IC-32314 (Oct. 13, 2016), 81 Fed. Reg. 81,870 (Nov. 18, 2016). At the same time, the Commission also amended Form N-PORT to require disclosure of certain information regarding the liquidity of a fund's holdings. Investment Company Liquidity Risk Management Programs, Release Nos. 33-10233, IC-32315 (Oct. 13, 2016), 81 Fed. Reg. 82,142 (Nov. 18, 2016). For more information regarding these rulemakings, including Form N-PORT requirements, please refer to Fund Alert, "Investment Company Reporting Modernization Amendments – A Summary of an Extensive Overhaul" (Nov. 7, 2016); Fund Alert, "What You Need to Know About the SEC's New Liquidity Risk Management Rule" (Nov. 2, 2016).
8 See Rule 30b1-9.
9 General Instruction F to Form N-PORT.
10 Release, supra note 1, at 58,732 – 33.
11 Chairman Jay Clayton, Statement on Cybersecurity (Sept. 20, 2017).
12 See Release, supra note 1, at 58,733; Investment Company Reporting Modernization, supra note 5, at 81,908.
13 Release, supra note 1, at 58,733.
14 Id.
15 Portfolio information attached as exhibits to Form N-PORT for the first and third quarters of a fund's fiscal year was to be made public during this period, to ensure that information about funds' portfolio holdings would continue to be publicly available to investors and other users. This practice will not change.
16 The Release states that reports for the periods ending March 31, 2019, through Sept. 30, 2019, will be kept nonpublic to preserve the six-month nonpublic period. Release, supra note 1, at 58734. However, this would be a seven-month period. We believe the Commission may have intended that the nonpublic period would end with (i.e., not include) the report for the period ending Sept. 30, 2019.
17 Form N-CEN, which requires annual reports of census-type information in a structured data format, was adopted in the same rulemaking as Form N-PORT.
18 See Investment Company Liquidity Risk Management Programs, supra note 5.
19 In the case of larger fund groups, which previously were required to begin filing liquidity information on Form N-PORT no later than Jan. 30, 2019, there will be only a three-month period before they have to begin making filings with liquidity information, by April 30, 2019. However, they will also have the benefit of the six-month period during which reports will not be publicly disclosed.
20 Release, supra note 1, at 58,733 n.25.
21 See Letter from Dorothy Donohue, Acting General Counsel, ICI, to Brent J. Fields, Secretary, Securities and Exchange Commission (Nov. 3, 2017); Letter from Timothy W. Cameron, Head, and Lindsey W. Keljo, Managing Director and Associate General Counsel, SIFMA AMG, to Chairman Jay Clayton, Commissioner Kara Stein, and Commissioner Michael Piwowar, Securities and Exchange Commission (Sept. 12, 2017).
Michael E. Schapiro
Fund Alert, March 17, 2017
SEC Enforcement Developments of Note for Mutual Funds and Their Advisers:
The Year in Review and a Look Ahead
Fund Alert, March 7, 2017
Section 36(b) Plaintiffs' 'Retained Fee' Theory Rejected in Kasilag Verdict for Defendants
Fund Alert, November 7, 2016
Investment Company Reporting Modernization Amendments – A Summary of an Extensive Overhaul
What You Need to Know About the SEC's New Liquidity Risk Management Rule
Fund Alert, October 24, 2016
What You Need to Know About the SEC's New Swing Pricing Rule
Fund Alert, September 2016
Court Rules for Defendants in Excessive Fee Case After Full Trial
Meet The Team Real Estate

redpajama_set_name: RedPajamaCommonCrawl


The Host Committee for the Michigan CBC Delegation Reception & Motown Live Afterglow requests the honor of your presence at its signature events, during the Congressional Black Caucus Foundation's Annual Legislative Conference in Washington, D.C.
*PLEASE NOTE: Michigan CBC Delegation Reception tickets include gourmet cuisine and dessert bar, drink promotion, featured artist performance and Motown Live Afterglow. In addition to these amenities, VIP TICKET HOLDERS will receive expedited admission, access to reserved seating (first come, first served) and entry into VIP Sponsors Lounge, including complimentary guest bar.
**PLEASE NOTE: Guests with "AFTERGLOW ONLY" tickets will not be admitted into the venue until 10:00 PM. Tickets include access to featured artist and celebrity deejay performance. While guests will have access to gourmet dessert bar, food will not be available during the Motown Live Afterglow.


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Daughter says father made threats day before fatal shootings
APPLING, Ga. - The daughter of a northeast Georgia man suspected of shooting five people to death before killing himself says her father was a "ticking time bomb."
Lauren Hawes told The Associated Press on Saturday that she, her mother Angela Dent and her 1-year-old daughter hid in a neighbor's house -- barely escaping with their lives -- while her father, Wayne Anthony Hawes, 50, went on a bloody rampage and killed five people, including her grandmother and cousin.
"He made threats before, but we never thought it would be at this capacity," Lauren Hawes said. "He's been kind of a ticking time bomb if you want to put in a few words."
Capt. Andy Shedd of the Columbia County Sheriff's Office said in a statement that the Friday night shootings stemmed from a domestic dispute that left three men and two women dead at two separate locations within about a mile of each other. The body of shooting suspect Hawes was recovered Saturday by authorities in his home in Appling.
Lauren Hawes, 26, confirmed that the bloodshed was connected to a domestic dispute between her parents: her mother had walked out on her father just a week ago. Angela Dent had left before -- but this time, she took her possessions with her to prevent Hawes from destroying them as he had done in the past.
After Dent's departure, Wayne Hawes bottomed out emotionally.
"He's done things that were questionable in the past, but never to this extent. This is very surprising. We thought he could possibly hurt himself, but not others," said Lauren Hawes.
The rampage began Friday evening, when sheriff deputies responded to a home at about 8 p.m. and found three victims. Authorities then were called to a second home nearby, where two other victims were found.
"We believe the two shootings were related based on witness accounts," Shedd said. When authorities reached Hawes' house and entered, they found him dead of a self-inflicted gunshot wound to the head. They also found evidence that he attempted to set the house on fire.
The victims were identified as Roosevelt Burns, 75; Rheba Mae Dent, 85; Trequila Clark, 31; Lizzy Williams, 59; and her husband Shelly Williams, 62. One of the female victims died on the way to the hospital, Shedd said. The others were dead at the scene.
"We believe some of the victims were related to the suspect's wife," Shedd said.
Lauren Hawes said her parents had known each since they were teenagers, and had a common law marriage.
Lauren Hawes said Rheba Mae Dent was her grandmother, and her cousin was Trequila Clark. She said her grandmother was retired and her cousin was a registered nurse, who graduated from Augusta State University in 2012. She said Roosevelt Burns was her grandmother's brother. She also said family knew the Williams family from church and while her dad wasn't much of a church-goer, he was neighborhood friends with Shelly Williams.
Ola Murry of Appling in northeast Georgia said the neighborhood is still devastated by the events. Murray said she thought Hawes was a nice guy, but he made a "stupid" decision.
She would see him around the neighborhood and he would often say hello while passing by.
"I always thought he was a nice guy," Murray said. "I know he did what he did, but that doesn't make him a bad guy. You know, the devil gets into you sometimes and you do stupid stuff. You got to think. You always have to put the Lord in front of you, let him lead you and you won't go wrong."
An investigation is ongoing.
Surveillance camera captures brazen daytime shooting in Southeast DC
Late firefighter's newborn daughter has moving photo shoot with dad's firehouse brothers


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Dyed and Gone to Heaven – An Online Magazine and Needlework Resource
The Stitched Pieces of Raymond Endicott MD
This month's Feature/Gallery had its genesis with an intriguing e-mail: "I just found out about a man who is a former radiologist from Seattle who developed post polio syndrome and then was diagnosed with MS. He now lives in Hawaii and is partially confined to a wheelchair. Despite this, he does an enormous amount of stitching, favoring complicated florals and Oriental designs and is by all accounts a superb stitcher."
Freesia in Brass Pot
by Leigh Designs
The gentleman in question turned out to be Raymond Endicott MD. And not only is Dr. Ray an ace stitcher, but he can cook up a gourmet meal and plays a "mean" piano to boot! Along with all this, he maintains a very unique and wry sense of humor. When presented with the very direct request that we wanted him for the Caron Collection website, he promptly responded, "I want you TOO!!" All in all, Dr. Ray is one "hell-of-a" guy!
Mother & Daughter on Bridge
By Toni's Needlepoint Designs
Dr Ray has never been one to let adversity keep him down. In his own words, his story unfolds, "I suffered Bulbar and Spinal Poliomyelitis at age of 14 in 1949 and spent one year recovering and undergoing rehabilitation. I graduated Medical School, University of Oregon in 1961.
by Lee's Needle Arts
During my internship I was on the Obstetric service and noticed that several of the Doctors did needlepoint while waiting to deliver babies. I became interested and they taught me how over the next 3 months. I have been needlepointing ever since.
It was always funny when I should fly to meetings. There I was in a three-piece business suit, doing needlepoint while other people read or did crosswords. I always got the strange looks and was always the subject of conversation. Rosy Greer went through the same thing, I believe."
Dr Ray practiced Neuro-Radiology and then Diagnostic Radiology until 1985, when he was diagnosed with Post Polio Syndrome. This is a progressive muscular atrophy secondary to his having had Polio in 1949. Five years later he was also diagnosed with Multiple Sclerosis, which in retrospect he has had since 1973.
Song of Mississippi Jolity
Tiger Sun Orchid Hamburen Orchid
The orchid designs above (there are a total of six which Dr. Ray has completed) will be framed in koa wood, in shadow boxes with two frames holding three each. He will hang them on his lanai. Dr Ray adds, "At the time of the Orchids I was using mostly Impressions and Metallics. In my new 'period' (I sound like Picasso) I have been getting into Watercolours, Waterlilies and all of the other new fibers."
Dr. Ray continues, "With these two diseases I was forced to retire. My wife and I moved to Kona, Hawaii in 1988 and I have been doing quite well since that time. I can move around the house. However, I go by wheelchair everywhere else."
#KB02
(Kimono)
Dr Ray engages in stitching Petite Point at least 5 hours a day while tuning into the "Soaps!" He states emphatically, "I Petite Point only to keep or give away." The shop that keeps Dr Ray well supplied with all his stitching needs is Lori's Needle Ark in Coeur d'Alene, ID, our featured Shop Focus this month. Dr Ray says of the shop's owner, "Lori Elmore and I are great friends and have lots of fun with new fibers and new canvases. Lori's mother was a second mother to me as I grew up in Spokane."
Lady with Dove
by Evelyn Bernstein Studio
Lori says, "When I started working with him [Dr. Ray] he did only European canvases and tapestry wool. Now he works with a variety of fibers on painted canvases. He sent me off learning new fibers when he started to lose the nerves between his fingers and found it very difficult to lay fibers. Recently I 'wised up' and sent him some color cards so he can choose some of his fibers. He also has Stitches for Effect and is learning how to incorporate different stitches."
This orchid design was the first hand-painted one that Lori sent to Dr. Ray. It employs a variety of fibers: silk, flair, metallics, etc. Lori recalls, "He said at the time that I had spoiled him."
What is Dr. Ray currently working on? Lori clues us in, "He is now working on Monet's Waterlilies from Magic Needle and a very large canvas from Lee's Needle Arts of a Japanese lady standing in front of an antique flower cart. The background will again be Watercolours, Hint of Mint. He says it really makes it look like a watercolor."
Foo Dog
As mentioned earlier, Dr. Ray is a gourmet cook and also bakes his own bread. He plays, in his own words, "a mean" piano (piano bar type music). Another example of his "playful" sense of humor came through when asked how he was coming along with his notes for this feature. He duly reported, "I am working on my Bio and shall have it out tomorrow. Have faith and if that doesn't work have another Egg Nog!"
We wish to thank Dr. Ray for his participation and most especially for his candor. One truly need look no further for inspiration!
Raymond Endicott M.D.
E-Mail: ORCIDRAYGERTa@webtv.net
Aloha Ke Akua
Note: All designs shown are available from:
Lori's Needle Ark
123 Sherman Ave
Coeur d' Alene, ID 83814
E-mail: lorinark@icehouse.net
COPYRIGHT NOTICE: No part of this feature story nor the included designs can be reproduced or distributed in any form (including electronic) or used as a teaching tool without the prior written permission of the CARON Collection Ltd. or the featured designers or contributors.
© 1997 The Caron Collection Ltd. / Voice: (203) 381-9999, Fax: 203 381-9003
CARON email: mail@caron-net.com / Webmaster monika@nika-net.com


Immaculate Classic French Country style home with many upgrades. As you pass through the elegant front door with intricate glass work & wrought iron handles you are greeted with 14-foot ceilings, cascading arches & spacious living areas basked in natural light & adorned with hardwood trim. 4 bedroom, 3 bath plus a bonus room and the Master Suite includes tray ceilings, large walk in shower, 2 shower heads & plenty of storage space. This beautiful home is complimented with a large back yard with updated landscaping & a Red Wood pergola with textured slab, beams & clear cover to filter light & cooler temperatures. This hard to find gem is loaded with character & will not last. Fireplace logs do not convey.
Offered by Glenda Hall of Era Courtyard Real Estate.


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It has been proven time and time again that music has the power to transcend the bounds of society. Always on the side of the oppressed, it lives within the souls of those that cling to it like a heartbeat. It has transformed countries, sparked movements and inspired those who are open to its message.
MTV's show "Rebel Music" showcases youth around the world who fight against oppression through music. The most recent episode highlighted Frank Waln, Inez Jasper and a handful of other Native American singers/rappers from various tribes across the US and Canada. I'd definitely be lying if I said that I expected a show like this to come from MTV. Regardless, it's something that needs to be talked about widespread. Too many times the voices of the oppressed are stifled by those unwilling to hear the cries. The only thing that loves company more than misery is ignorance. The episode brought up so many important issues, particularly the negative effects the Keystone Pipeline would have on Native American lands, extremely high suicide rates among Native American youth and the disproportionately high number of indigenous women in Canada being murdered.
Watching this episode, it was sad but not surprising to hear that so many issues that Native Americans are facing are some of the very same issues are currently being protested across the country by the black community – police brutality, cultural appropriation, longstanding stereotypes that further perpetuate and sustain our oppressions. In likeness of Black Lives Matter, Native Americans are also seeking a voice in the conversation with #NativeLivesMatter.
I want to make it clear to anyone who feels as though focusing on both of these movements will diminish the other that that is not the case. I stand behind both movements because, while our cultures are different, our goals are the same. Our oppression binds us together, but it is only our solidarity with each other that can (and will) break the chains that colonialism has tried so hard to keep us in.
So I think it's time for a little shoutout. I started this blog a few months now, but, during that time, I've come across a handful of blogs that prove to be precious little gems in a sea of thoughts.
One of these gems is Media Diversified. It's a blog dedicated to diversifying the media landscape. The non-profit organization blog features dozens of writers and academics from all walks of life, from all parts of the world. It's refreshing that they don't shy away from any and all topics that interest them – from oppressions of people of color in films to the exploration of Afrofuturism.
I will say that I'm slightly disappointed that they are based in the UK. A blog like this would NO DOUBT have such a positive reception among the ranks of activists and feminists across the US, especially in light of the numerous police brutality videos that have been flooding social media with the help of the American people. With a blog similar to this, the US could definitely see more organized protests and leaders emerge from the depths.
Once you browse through their blog, you'll definitely want to reblog and share A LOT of the articles you come across. However, they do have reposting guidelines that you'll want to look over before reposting any of their work.
Last Thursday, Republican Governor Mike Pence signed the Religious Freedom Restoration Act into law in Indiana. The law potentially allows for-profit businesses to refuse service to certain groups based on any religious grievances these businesses might encounter. To understand why this law is seeded in so much controversy, let's look at its origins.
The Religious Freedom Restoration Act (RFRA) was first signed into law by Bill Clinton in 1993. While the bill applies to all religions, its original creation was to protect both the sacred lands of Native Americans from being destroyed by US expansion projects and the use of peyote for Native American religious ceremonies. Since then, numerous states have enacted laws that mirror their federal predecessor.
The main reason this law is so dangerous is the potential increase of discrimination that could arise from its enactment. Though there are cities that have nondiscrimination laws, statewide Indiana does not have laws that protect those who identify as LGBTQ. Ultimately, this simply means that the RFRA leaves the door wide open for prejudice and bigotry against a group of people who are already unprotected against discrimination.
As I previously stated, the original purpose of this act was to safeguard against discrimination of Native American tribes and their religious practices. Despite this, I highly doubt that this is the case at hand. Yes, I do understand that religion is deeply engrained in the lives of many Americans. I also understand why people want to protect these religious views. What I am not grasping, however, is why Mike Pence and other supporters of this law feel that their religious rights are being infringed upon by having to provide service to certain groups of people. Mike Pence and other proponents of the RFRA in Indiana most likely prescribe to Christian values. Because of this, they automatically belong to the religious majority, which, in essence, allows them religious privilege. Christians do not have to fight for our religious rights like Muslims, Buddhists, Native Americans or other religious minorities might. Would this law be so accommodating as to protect a Muslim's right to refuse Christian customers on the basis of "substantial burden?"…I highly doubt it. And what about individuals who identify as BOTH Christian AND LGBTQ? Will this law still allow for-profit businesses to refuse services to them as well?
So, what has people so up in arms about the implications of this law? To anyone who has been keeping up with the Indiana RFRA law, the answer is simple. Honestly, I would have no problem if this specific bill was created to protect the rights of religious minorities. But when Mike Pence dodges George Stephanopoulos' repeatedly asked question, the intent of this law could not be clearer. For someone wanting to clarify the "misunderstanding" of this law, Pence did an amazing job at doing the complete opposite. While Pence voiced that the law would not target LGBTQ, anti-gay activists were present at the signing last Thursday.
Thousands of Americans have responded to the law, some of which believe boycotting Indiana would be the wisest choice. While boycotting Indiana certainly brings attention of the RFRA law to the forefront of conversation regarding LGBTQ discrimination, I believe it will hurt hundreds of Indiana businesses that don't agree with the bill. For those Indiana businesses whose views don't align with the recent law, the campaign Open For Service provides participating business with a sticker to illustrate their solidarity and commitment to opposing discrimination of any kind. Though a fairly new grassroots campaign, it's a step in the right direction. Others have taken to social media to voice their opposition to the new law, particularly with the hashtag #DearMikePence.
More respect could – and would- be given to Pence if he stood firm in his beliefs. But to shamelessly masquerade them under the guise of religious protection is insulting to all parties involved.
Regardless of how people are choosing to act out against Indiana's RFRA, one thing is still clear: this law is a dangerous obstacle in the fight for equality.


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A More Efficient Way to Make Induced Pluripotent Stam cells
Mark Stadtfeld and his colleagues at the NYU Longone Medical Center has devised a new method for making induced pluripotent stem cells that greatly increases efficiency at which these cells are made.
Induced pluripotent stem cells or iPSCs are made from mature, adult cells by mean of a combination of genetic engineering and cell culture techniques. In short, the expression of four genes is forced in adult cells; Oct4, Sox2, Klf4, and c-Myc or OSKM. The proteins encoded by these four genes cooperatively work to drive a fraction of the cells into an immature state that resembles that of embryonic stem cells. These cells are them grown in cell culture systems that select for those cells that can grow continuously and form colonies of cells derived from progenitor cells. These cell colonies are them repeated isolated a re-cultured until an iPSC line has been established.
Unfortunately, this process is rather inefficient and tedious, since less than one percent or so of the reprogrammed cells actually undergo successful reprogramming. Additionally, it can take several weeks to properly establish an iPSC line. Thus, stem cell scientists have been looking at several different ways to boost the efficiency of this process.
Stadtfeld and his coworkers tried to add compounds to the cultured cells to determine if the culture conditions could actually augment the efficiency of the reprogramming process. "We especially wanted to know if these compounds could be combined to obtain stem cells at high-efficiency," said Stadtfeld.
The compounds to which Stadtfeld was referring were two cell signaling proteins called Wnt and TFG-beta. Both of these compounds regulate a host of cell growth processes. Stadtfeld wanted to try regulating both of these pathways at the same time, in addition to providing cells with ascorbic acid, which is also known as vitamin C. Even vitamin C is more popularly known as an antioxidant, vitamin C also can remodel chromatin (that tight structure into which cells package their DNA).
When mouse skin fibroblasts were treated with OSKM and a compound that activates Wnt signaling, the efficiency of iPSC derivation increased slightly. The same thing was observed if fibroblasts were treated with OSKM and a compound that inhibits TGF-beta signaling or vitamin C. However, when all three of these compounds were combined, OSKM-engineered fibroblasts were reprogrammed at an efficiency of close to 80 percent. When different cell types were used as the starting cell, such as blood progenitor cells, the efficiency jumped to close to 100 percent; a result that was also observed if liver progenitor cells were used as the starting cell.
Stadtfeld is confident that these dramatic increases in iPSC derivation should improve future studies with iPSCs, since his protocol should make iPSC derivation more predictable. "It's just a lot easier this way to study the mechanisms that govern reprogramming, as well as detect any undesired features that might develop in iPSCs," he said.
Vitamin C and the two compounds used to manipulate the Wnt and TGF-β pathways have been widely used in research and have few unknown or hazardous effects. However, OKSM has in some cases caused undesired features in iPSCs, such as increased mutation rates. Stadtfeld believes that by making iPSC induction more rapid and efficient, his new technique might also make the resulting stem cells safer. "Conceivably it reduces the risk of abnormalities by smoothening out the reprogramming process," Dr. Stadtfeld says. "That's one of the issues we're following up."
Posted on October 23, 2014 October 23, 2014 Categories Alternatives to Embryonic Stem Cells, Induced Pluripotent Stem Cells, Model Systems, Stem Cell BiologyTags ascorbic acid, TGF-beta1 signaling, vitamin C, Wnt signaling


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May's New Releases: Games
Even though we're approaching the notoriously slow summer months, the UK games release schedule is still looking pretty great! Check out the new game releases this month below.
This prequel to Wolfenstein: The New Order began life as downloadable content but was eventually transformed to a game in its own right. The Old Blood follows William 'B.J' Blazkowicz as he attempts to break into Castle Wolfenstein, before meeting a Nazi archaeologist who's looking for some evil-unleashing artifacts.
Kirby and the Rainbow Paintbrush
It's been a tough couple of years for the Wii U, but Nintendo will be hoping Kirby and the Rainbow Paintbrush is the start of a grand comeback. It certainly uses the Wii's gamepad to good effect, with players drawing on it to guide Kirby through the various levels.
Kirby and the Rainbow Paintbrush has been out for a couple of months overseas already, and reviews have been mixed. That said, this should be worth it for younger gamers and Wii U owners looking for something new.
Originally set for release in November 2014, this sandbox racing game – which sees players take up a career in motorsport, tackling various disciplines from karting to full-on supercar racing – has been delayed numerous times. This time, though, we're fairly certain it'll be coming out. And it looks pretty decent too!
Codename S.T.E.A.M
This turn-based strategy for the 3DS features the vocal talents of Wil Wheaton as…erm, Abraham Lincoln battling an alien invasion. It looks every bit as wacky as it sounds.
CD Projekt's RPG series is so popular that President Barack Obama namechecked it in a speech. Here's hoping Wild Hunt, which follows hero Geralt as he tries to stop a supernatural invasion, matches those lofty standards. Apparently, it's 20 times bigger than Skyrim, so we're on the right track.
Farming Simulator '15
According to Wikipedia, Farming Simulator is 'the most widely played realistic farming simulation game' in the world. Who are we to disagree?
In case you aren't familiar, Farming Simulator tasks you with running a farm: you'll have to plant crops, commandeer tractors (there's over 41 brands to choose from) and run a profitable business. This new version introduces foresty and vehicle washing into the mix. Trust us, it's worryingly addictive.
It's not often that Nintendo unveil a game without any of their main characters, so Splatoon is a bit special. It's a third person shooter in the style of Gears of War, but, true to form, Nintendo have replaced gruff space marines with colourful teenagers and chainsaws with paintball weapons. Oh, and you can turn into a squid too. Yep, this is going to be special.
If you want to buy any of this month's new releases, why not sell games with musicMagpie and make a bit of cash? Just click the button below to get started.
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Please look out for Foam From Above's posts from Toy Fair 2018. He's got the first appointment for Hasbro and NERF and I'll be really excited to see what he tries out with some of these blasters.
I definitely want to get in on that Infinus action, but I am unsure about the rest for me personally. I'm still thinking about the Nemesis when it comes to Rival power and mag/hopper size!
Let me know what you think about the latest announcement. Please also consider subscribing to the Youtube channel. We've preserved partner status so far, but I would love to solidify that going into our new series this Spring!
Click Click BAMF Rises Again!
That new video is up that I promised. It's kind of long for a straight talking piece, so there are timecodes in the description for where I talk about what.
As you see, we just had to talk a little bit about the Youtube Partnership complaints. We appreciate all the support that fans are showing the community. It's just time to realize that Google isn't going to hand out prizes for existing. That's over and it's okay!
I don't know if you've seen the new press releases for the latest two Nerf announcements, but they're not bad! Foam From Above posted them and I haven't been thinking about the Infinus' dart loading system. I want to see this "motorized Quick-Load Technology; as kids feed darts into the blaster, the darts are automatically loaded into the 30-dart removable drum." My main worry is the potential for movable parts to get broken by human error or normal use. But I just have to check it out as soon as we can!
Systems restarting. Please do not adjust your monitors . . . .
But seriously, guys. We are really looking at jumping back in to the Nerf scene in a different way. Let's just say gameplay is and always will be the best part of the hobby. Looking to bring you some video that isn't just someone's GoPro footage of a war they went to or overdone beauty shots from a glidecam.
Expect a new Youtube video sometime this week about several things. Why CCB went dormant. Youtube Partnership and why folks should quit whining. More importantly; info will soon be coming about plans for a new pilot we're hoping to debut on the Click Click BAMF Youtube Channel some time this Spring.
In the mean time, check out Foam From Above gearing up for the Toy Fair 2018! As always, I am super-stoked to see what he finds out.
Thanks for your continued support over these last two years. It means more than you could ever know.


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PDP group urges calm over court ruling on Anambra chair
Independent Leaders Forum (ILF) has called for calm over the ruling of the Federal High Court, Abuja, which on Wednesday returned Ejike Oguebego as the chairman of the party in Anambra State.
This is just as Chief Ndubuisi Nwobu, who had been functioning as chairman of the party before the latest ruling, dismissed the judgment as political coup. ILF is a pressure group in the PDP.
In a statement issued in Enugu yesterday, Secretary of ILF, Frank Molokwu, asked party faithful to remain calm until the party receives the Certified True Copy (CTC) of the judgment.
He said: "It is rather unfortunate that these individuals have continued to exhibit same antics against the party, as they have done for 18 years, without exhausting the internal party mechanism on conflict resolution. We, therefore, consider this latest judgment as an affront and threat to the growth of the younger politicians and progress of the party in the state."
He expressed his group's intention to appeal the ruling.
HOWEVER, Anambra PDP has said there is no cause for alarm as its executive team is still intact and operational.
Nwobu dismissed the ruling that set aside the judgment of Justice D. Musa, describing it as a coup d'état in a political space. He recalled that Justice Musa had declared the Anambra PDP caretaker committee led by Chukwudi Umeaba as null and void, having been constituted in breach of the extant provisions of the party.
My directive to shoot criminals with AK-47 stands – Buhari
Lagos begins rehabilitation of Rowe Park
Young boys spray money lavishly in a church while being hailed by members (video)
Gossip, Lifestyle
Atletico Madrid win LaLiga Santander 2020/21 title (photos)
News, sports
Popular Nigerian lesbian, Amara, denied an apartment after landlady went through her social media handles and discovered she is a lesbian
"Rose, rest na, no one is fighting man with you" – Tboss's sister, Goldie tells Rosy Meurer


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New ASIS Foundation Research Explores the State of Security Convergence
Andy Cutler
pr@asisonline.org
Only 24% of study respondents have converged their physical and cybersecurity functions
Alexandria, VA (18 December 2019)—ASIS International, the world's largest association of security management professionals, is pleased to announce it has published new State of Security Convergence in the United States, Europe, and India research conducted by the ASIS Foundation and sponsored by AlertEnterprise.
Using survey responses from more than 1,000 security leaders from around the globe—plus more than 20 follow-up interviews—the study analyzes the relationship between physical security, cybersecurity, and business continuity in modern organizations. It provides relevant benchmarks to compare strategies, plans, and operations and determine best practices for creating more effective and cost-efficient security and risk operations.
According to the report, despite years of predictions about the inevitability of security convergence, just 24 percent of respondents have converged their physical and cybersecurity functions. When business continuity is included, a total of 52 percent have converged two or all of the three functions. Of the 48 percent who have not converged at all, 70 percent have no current plans to converge.
"For years, security practitioners have accepted that organizations are increasingly converging their physical security and cybersecurity functions," says Brian Allen, CPP, president, ASIS Foundation Board of Trustees. "This study collected current data to measure trends and progress with converging environments. What we've learned is that, although convergence has brought positive results, there is still much work to be done."
The study's executive summary is available free on the Foundation's website. The full report is available for purchase and is complimentary for all ASIS members.
Supported by member and corporate donations, the ASIS Foundation invests in elevating security practice through research and education. The Foundation awarded more than 170 scholarships in 2019 totaling more than $75,000. Learn more about 2020 ASIS Foundation scholarships.
About the ASIS Foundation
The ASIS Foundation, a 501(c)(3) nonprofit organization, elevates the security profession worldwide through research and education. The Foundation awards grants and scholarships to help chapters and individuals, including those transitioning to careers in security management, achieve their professional and academic goals. Governed by a Board of Trustees, the Foundation is supported by generous donations from individuals, allied organizations, ASIS chapters and councils and ASIS International. To learn more, visit www.asisfoundation.org.
About AlertEnterprise
In today's threat landscape, operating with siloed physical security, IT and cyber systems puts your enterprise at greater risk. Cyber and physical threats are now blended, requiring a converged approach that fully integrates and automates security with operations and compliance. At AlertEnterprise we bring people, processes, data and technology together in a way which increases daily intelligence and reduces risk. That's what we call security convergence and it's our daily mission. With our trusted identity and access management platform, enterprises can do more with less, create engaging workforce experiences, increase compliance and mitigate threats and risk. For more information, visit www.alertenterprise.com.
About ASIS International
Founded in 1955, ASIS International is the world's largest membership organization for security management professionals. With hundreds of chapters across the globe, ASIS is recognized as the premier source for learning, networking, standards, and research. Through its board certifications, award-winning Security Management magazine, and Global Security Exchange (formerly the Annual Seminar and Exhibits)—the most influential event in the profession—ASIS ensures its members and the security community have access to the intelligence and resources necessary to protect their people, property, and information assets. Learn more about the work we do at www.asisonline.org.