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Cervical spontaneous spinal epidural hematoma (CSSEH) is a rare condition that can be potentially fatal if not properly diagnosed and managed. While prompt surgical decompression and evacuation of the hematoma are generally considered as the first line of treatment, mild cases that were managed through observation and conservative treatment have been reported. Our patient was a 24-year-old man who experienced two CSSEH events 8 months apart, both of which were managed conservatively. This was a rare case of recurrent CSSEH in which recovery was achieved without surgical intervention. We believe conservative treatment with close observation may be effective in CSSEH patients presenting with mild neurologic symptoms who have a tendency towards spontaneous neurologic improvement.

Objective: The ossification of the ligamentum flavum (OLF) is one of the major causes of thoracic myelopathy. Surgical decompression with or without instrumented fusion is the mainstay of treatment. However, few studies have reported on the added effect of instrumented fusion. The objective of this study was to compare clinical and radiological outcomes between surgical decompression without instrumented fusion (D-group) and that with instrumented fusion (F-group).Methods: A retrospective review was performed on 28 patients (D-group, n=17; F-group, n=11) with thoracic myelopathy due to OLF. The clinical parameters compared included scores of the Japanese Orthopedic Association (JOA), the Visual analogue scale of the back and leg (VAS-B and VAS-L), and the Korean version of the Oswestry disability index (K-ODI). Radiological parameters included the sagittal vertical axis (SVA), the pelvic tilt (PT), the sacral slope (SS), the thoracic kyphosis angle (TKA), the segmental kyphosis angle (SKA) at the operated level, and the lumbar lordosis angle (LLA; a negative value implying lordosis). These parameters were measured preoperatively, 1 year postoperatively, and 2 years postoperatively, and were compared with a linear mixed model.Results: After surgery, all clinical parameters were significantly improved in both groups, while VAS-L was more improved in the Fgroup than in the D-group (-3.4±2.5 vs. -1.3±2.2, p=0.008). Radiological outcomes were significantly different in terms of changes in TKA, SKA, and LLA. Changes in TKA, SKA, and LLA were 2.3°±4.7°, -0.1°±1.4°, and -1.3°±5.6° in the F-group, which were significantly lower than 6.8°±6.1°, 3.0°±2.8°, and 2.2°±5.3° in the D-group, respectively (p=0.013, p

Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear. Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury. Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes. Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.

C2–3 disc herniation is rare and a definitive treatment of choice has not been established. The purpose of this case report is to suggest posterior approach as one of the best options. A 49-year-old man visited our clinic with a 7-year history of neck pain and occipital headache and a 2-month history of right arm pain. C2–3 intervertebral disc herniation of the central type was diagnosed on magnetic resonance imaging (MRI), and surgery was performed, including C1 laminectomy, C2–3 laminoplasty, and C2–3 posterior fixation. The posterior approach was used because the patient's neck was difficult to operate anteriorly. After 3 months postoperatively, MRI showed widened cerebrospinal fluid space at the C2–3 level. The visual analogue scale score for pain improved in the occipital area and right arm. However, the untouched protruded central disc, subjective weakness in right hand grasping, and numbness persisted. In conclusion, this case highlights posterior decompression and fixation as a good treatment of choice for decompression at the C2–3 level disc herniation, from where it is difficult to remove compressive lesions directly via the anterior corridor.

SPAK [STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase] kinase consists of a full-length (FL-) and an alternatively spliced kidney-specific (KS-) isoform. SPAK regulates the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). The relative abundance and role of FL- vs. KS-SPAK in regulating Na+-K+-2Cl− cotransporter (NKCC2) in thick ascending limb (TAL) are not completely understood. Here, we report that FL-SPAK mRNA was the most abundant in medullary TAL (mTAL), followed by cortical TAL (cTAL) and DCT. KS-SPAK mRNA abundance was relatively lower than FL-SPAK. The ratios of FL-SPAK to KS-SPAK in mTAL, cTAL, and DCT were 12.3, 12.5, and 10.2, respectively. To examine the role of SPAK in the regulation of sodium transport in TAL, we used in vitro microperfusion of mTAL and cTAL isolated from wild-type (WT) and SPAK knockout mice (SPAK-KO) that lack both FL- and KS-SPAK. The rates of sodium absorption in cTAL and mTAL of SPAK-KO mice were 34.5 and 12.5% of WT tubules, respectively. The mRNA levels of related OSR1 kinase and SPAK protease Dnpep in SPAK-KO tubules were not significantly different from WT tubules. We next examined the role of SPAK in the regulation of sodium reabsorption by vasopressin in TAL. Vasopressin increased sodium reabsorption by ∼80% in both mTAL and cTAL from WT mice. While baseline sodium reabsorption was lower in SPAK-KO tubules, vasopressin increased sodium reabsorption over twofold. In conclusion, the combined net effect of SPAK isoforms on sodium reabsorption in TAL is stimulatory. SPAK is not essential for vasopressin stimulation of sodium reabsorption in TAL.

e22510 Background: GCT is a benign and locally aggressive tumor that occur infrequently in the mobile spine. The radical excision is generally agreed for treatment of GCTs. Methods: We performed retrospective analysis of 20 GCTs in the mobile spine from 2005 to 2013. Verteberal involvement was classified with Weinstein-Boriani-Biagini grading system. Surgery types were classified into 3 groups; partial excision, gross total removal (GTR), and en-bloc resection. We analysis by GTR versus non-GTR group, and enbloc versus non-enbloc group in GTR group. The log rank test was used for comparison of factors, and a P value of less than 0.05 was deemed significant. Results: After first surgery of the 20 patients, 17 additional surgery performed for 10 (50%) patients. There were 7 (35%) local recurrences and 3 (15%) distant metastasis. The time to recurrence or progression of tumor ranged from 5 months to 39 months. Two (10%) patients had additional surgery for instrument failure. One (5%) patient died from the complication of the surgery. The follow up duration ranged from 24 months to 141 months. The local recurrence was associated with partial resection (P < 0.01) and history of recurrence (P = 0.39). There was no significant difference between non-en bloc GTR and en bloc GTR in local recurrence (p = 0.101). Conclusions: Gross total resection should be considered for GCTs of the mobile spine. The choice of en bloc resection must be balanced with risks of the procedure. En bloc resection or radical excision could be challenging in many cases. In that cases, the gross total removal by intralesional resection is considerable for local control.

Soluble Klotho (sKlotho) is the shed ectodomain of antiaging membrane Klotho that contains 2 extracellular domains KL1 and KL2, each of which shares sequence homology to glycosyl hydrolases. sKlotho elicits pleiotropic cellular responses with a poorly understood mechanism of action. Notably, in injury settings, sKlotho confers cardiac and renal protection by down-regulating calcium-permeable transient receptor potential canonical type isoform 6 (TRPC6) channels in cardiomyocytes and glomerular podocytes. Inhibition of PI3K-dependent exocytosis of TRPC6 is thought to be the underlying mechanism, and recent studies showed that sKlotho interacts with α2-3-sialyllactose-containing gangliosides enriched in lipid rafts to inhibit raft-dependent PI3K signaling. However, the structural basis for binding and recognition of α2-3-sialyllactose by sKlotho is unknown. Using homology modeling followed by docking, we identified key protein residues in the KL1 domain that are likely involved in binding sialyllactose. Functional experiments based on the ability of Klotho to down-regulate TRPC6 channel activity confirm the importance of these residues. Furthermore, KL1 domain binds α2-3-sialyllactose, down-regulates TRPC6 channels, and exerts protection against stress-induced cardiac hypertrophy in mice. Our results support the notion that sialogangliosides and lipid rafts are membrane receptors for sKlotho and that the KL1 domain is sufficient for the tested biologic activities. These findings can help guide the design of a simpler Klotho mimetic.-Wright, J. D., An, S.-W., Xie, J., Yoon, J., Nischan, N., Kohler, J. J., Oliver, N., Lim, C., Huang, C.-L. Modeled structural basis for the recognition of α2-3-sialyllactose by soluble Klotho.

Fil: Dalton, George. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Fil: An, Sung-Wan. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Fil: Al-Juboori, Saif I. University of Colorado Denver. Department of Electrical Engineering; Estados Unidos Fil: Nischan, Nicole. University of Texas Southwestern Medical Center. Department of Biochemistry; Estados Unidos Fil: Yoon, Joonho. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Fil: Dobrinskikh, Evgenia. University of Colorado Denver. Department of Medicine; Estados Unidos Fil: Hilgemann, Donald W. University of Texas Southwestern Medical Center. Department of Physiology; Estados Unidos Fil: Xie, Jian. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Fil: Luby-Phelps, Kate. University of Texas Southwestern Medical Center. Department of Cell Biology; Estados Unidos Fil: Luby-Phelps, Kate. University of Texas Southwestern Medical Center. Live Cell Imaging Core Facility; Estados Unidos Fil: Kohler, Jennifer J. University of Texas Southwestern Medical Center. Department of Biochemistry; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Neurobiology Laboratory; Estados Unidos Fil: Huang, Chou-Long. University of Texas Southwestern Medical Center. Department of Medicine; Estados Unidos Abstract: Soluble klotho, the shed ectodomain of the antiaging membrane protein α-klotho, is a pleiotropic endocrine/paracrine factor with no known receptors and poorly understood mechanism of action. Soluble klotho down-regulates growth factor-driven PI3K signaling, contributing to extension of lifespan, cardioprotection, and tumor inhibition. Here we show that soluble klotho binds membrane lipid rafts. Klotho binding to rafts alters lipid organization, decreases membrane's propensity to form large ordered domains for endocytosis, and down-regulates raft-dependent PI3K/Akt signaling. We identify α2-3-sialyllactose present in the glycan of monosialogangliosides as targets of soluble klotho. α2-3-Sialyllactose is a common motif of glycans. To explain why klotho preferentially targets lipid rafts we show that clustering of gangliosides in lipid rafts is important. In vivo, raft-dependent PI3K signaling is up-regulated in klotho-deficient mouse hearts vs. wild-type hearts. Our results identify ganglioside-enriched lipid rafts to be receptors that mediate soluble klotho regulation of PI3K signaling. Targeting sialic acids may be a general mechanism for pleiotropic actions of soluble klotho.

Summary Background PLC-β signaling is generally thought to be mediated by allosteric activation by G proteins and Ca 2+ . Although availability of the phosphatidylinositol-4,5-biphosphate (PIP 2 ) substrate is limiting in some cases, its production has not been shown to be independently regulated as a signaling mechanism. WNK1 protein kinase is known to regulate ion homeostasis and cause hypertension when expression is increased by gene mutations. However, its signaling functions remain largely elusive. Results Using diacylglycerol-stimulated TRPC6 and inositol trisphosphate-mediated Ca 2+ transients as cellular biosensors, we show that WNK1 stimulates PLC-β signaling in cells by promoting the synthesis of PIP 2 via stimulation of phosphatidylinositol 4-kinase IIIα. WNK1 kinase activity is not required. Stimulation of PLC-β by WNK1 and by Gα q are synergistic; WNK1 activity is essential for regulation of PLC-β signaling by G q -coupled receptors, and basal input from G q is necessary for WNK1 signaling via PLC-β. WNK1 further amplifies PLC-β signaling when it is phosphorylated by Akt kinase in response to insulin-like growth factor. Conclusions WNK1 is a novel regulator of PLC-β that acts by controlling substrate availability. WNK1 thereby coordinates signaling between G protein and Akt kinase pathways. Because PIP 2 is itself a signaling molecule, regulation of PIP 2 synthesis by WNK1 also allows the cell to initiate PLC signaling while independently controlling the effects of PIP 2 on other targets. These findings describe a new signaling pathway for Akt-activating growth factors, a mechanism for G protein-growth factor crosstalk, and a means to independently control PLC signaling and PIP 2 availability.

Telomerase activation is a key step in the development of human cancers. Expression of the catalytic subunit, human telomerase reverse transcriptase (hTERT), represents the limiting factor for telomerase activity. In this study, we have used artificial zinc finger protein (ZFP) transcription factors (TF) to repress the expression of hTERT in human cancer cell lines at the transcriptional level. We have constructed four-fingered ZFPs derived from the human genome which binds 12-bp recognition sequences within the promoter of the hTERT gene and fused them with a KRAB repressor domain to create a potent transcriptional repressor. Luciferase activity was decreased by >80% in all of the transcriptional repressors with luciferase reporter assay. When they were transfected into the telomerase-positive HEK293 cell line, a decrease of mRNA level and telomerase activity together with shortening of telomere length was observed. Actual growth of HEK293 cells was also inhibited by transfection of artificial ZFP-TFs. The repression was maintained for 100 days of culture. The repression of telomerase expression by artificial ZFP-TFs targeting the promoter region of the hTERT presents a new promising strategy for inhibiting the growth of human cancer cells. Mol Cancer Res; 8(2); 246–53

We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.

Interferon-alpha (IFN-alpha) has been used to treat hepatitis C Virus (HCV)-induced infection but has been effective in only about half of all patients. It is suggested that the different responses to IFN-alpha treatment in HCV infection may be influenced by HCV genotypes, HCV RNA titer at the beginning of IFN-alpha therapy, and the sequences of the interferon sensitivity determining region (ISDR). However, there have also been reports showing that these have no relation to an IFN-alpha effect. In a previous study, it was found that the nucleotide sequence variation in the hypervariable region (HVR) 1 of the HCV could predict the effect of IFN-alpha. In the present investigation, an attempt was made to determine the predictive factors of IFN-alpha therapy. Twenty-six patients with HCV infection were treated with IFN-alpha. Among these, 13 patients recovered after 3 to 6 months of IFN-alpha treatment, although the other 13 patients showed no response after 6 months of treatment with IFN-alpha. In order to determine the predictive factors of IFN-alpha therapy, the ALT levels, HCV genotypes, HCV serum titer, and the quasispecies of HVR 1 were compared between responders and non-responders. It is suggested that the variation in the HVR 1 and HCV serum titer can be used to predict the effect of IFN-alpha.

Cardiac hypertrophy occurs in up to 95% of patients with CKD and increases their risk for cardiovascular death. In the kidney, full-length membranous Klotho forms the coreceptor for fibroblast growth factor 23 (FGF23) to regulate phosphate metabolism. The prevailing view is that the decreased level of Klotho in CKD causes cardiomyopathy through increases in serum FGF23 and/or phosphate levels. However, we reported recently that soluble Klotho protects against cardiac hypertrophy by inhibiting abnormal calcium signaling in the heart. Here, we tested whether this protective effect requires changes in FGF23 and/or phosphate levels. Heterozygous Klotho-deficient CKD mice exhibited aggravated cardiac hypertrophy compared with wild-type CKD mice. Cardiac magnetic resonance imaging studies revealed that Klotho-deficient CKD hearts had worse functional impairment than wild-type CKD hearts. Normalization of serum phosphate and FGF23 levels by dietary phosphate restriction did not abrogate the aggravated cardiac hypertrophy observed in Klotho-deficient CKD mice. Circulating levels of the cleaved soluble ectodomain of Klotho were lower in wild-type CKD mice than in control mice and even lower in Klotho-deficient CKD mice. Intravenous delivery of a transgene encoding soluble Klotho ameliorated cardiac hypertrophy in Klotho-deficient CKD mice. These results suggest that the decreased level of circulating soluble Klotho in CKD is an important cause of uremic cardiomyopathy independent of FGF23 and phosphate, opening new avenues for treatment of this disease.

WNK1 is a widely expressed serine/threonine protein kinase that regulates multiple cellular and organ functions via diverse mechanisms. We previously reported that endothelial-specific deletion of Wnk1 in mice results in embryonic lethality, with angiogenesis and cardiac defects beginning at embryonic day ∼10.5. Here, we further investigated the signaling mechanism by which WNK1 regulates embryonic cardiovascular development. We found that mice with a global deletion of Osr1, which encodes oxidative stress-responsive kinase-1, a protein kinase activated by WNK1, died in utero beginning at embryonic day ∼11. The defects in Osr1-null yolk sacs and embryos were virtually identical to those observed in Wnk1-knock-out mice: no mature large vessels in yolk sacs, defective angiogenesis in the brain and intersomitic vessels, and smaller chambers and reduced myocardial trabeculation in mutant hearts. Endothelial-specific deletion of Osr1 generated by crossing Osr1(flox/flox) mice with Tie2-Cre mice phenocopied defects caused by global Osr1 deletion. To investigate whether OSR1 acts downstream of WNK1 in embryonic angiogenesis, we generated a mouse line that carries a catalytically and constitutively active human OSR1 transgene in the ROSA26 locus under the control of a cassette of floxed transcription stop codons. We found that endothelial-specific expression of the constitutively active mutant OSR1, generated by Tie2-Cre-mediated excision of floxed stop codons in the mutated ROSA26 locus, rescued angiogenesis and cardiac defects in global Wnk1-null embryos. These results indicate that WNK1 activation of the OSR1 signaling cascade is an essential pathway that regulates angiogenesis and cardiac formation during mouse embryo development.

PLC-β signaling is generally thought to be mediated by allosteric activation by G proteins and Ca(2+). Although availability of the phosphatidylinositol-4,5-biphosphate (PIP(2)) substrate is limiting in some cases, its production has not been shown to be independently regulated as a signaling mechanism. WNK1 protein kinase is known to regulate ion homeostasis and cause hypertension when expression is increased by gene mutations. However, its signaling functions remain largely elusive.Using diacylglycerol-stimulated TRPC6 and inositol trisphosphate-mediated Ca(2+) transients as cellular biosensors, we show that WNK1 stimulates PLC-β signaling in cells by promoting the synthesis of PIP(2) via stimulation of phosphatidylinositol 4-kinase IIIα. WNK1 kinase activity is not required. Stimulation of PLC-β by WNK1 and by Gα(q) are synergistic; WNK1 activity is essential for regulation of PLC-β signaling by G(q)-coupled receptors, and basal input from G(q) is necessary for WNK1 signaling via PLC-β. WNK1 further amplifies PLC-β signaling when it is phosphorylated by Akt kinase in response to insulin-like growth factor.WNK1 is a novel regulator of PLC-β that acts by controlling substrate availability. WNK1 thereby coordinates signaling between G protein and Akt kinase pathways. Because PIP(2) is itself a signaling molecule, regulation of PIP(2) synthesis by WNK1 also allows the cell to initiate PLC signaling while independently controlling the effects of PIP(2) on other targets. These findings describe a new signaling pathway for Akt-activating growth factors, a mechanism for G protein-growth factor crosstalk, and a means to independently control PLC signaling and PIP(2) availability.

AIM: To confirm the predictive factors for interferon (IFN)-α and ribavirin combination therapy for chronic hepatitis patients with hepatitis C virus (HCV) genotype 1b. METHODS: HCV RNA from 50 patients infected with HCV genotype 1b was studied by cloning and sequencing of interferon sensitivity determining region (ISDR), PKR-eIF2α phosphorylation homology domain (PePHD). Patients were treated with IFN-α and ribavirin for 6 mo and grouped by effectiveness of the therapy. A variety of factors were analyzed. RESULTS: Our data showed that age, HCV RNA titer, and ISDR type could be used as the predictive factors for combined IFN-α and ribavirin efficacy. Characteristically, mutations in PePHD appeared only when the combination therapy was effective. Other factors, such as sex and alanine aminotransferase (ALT) level, were not related to its efficacy. Adjusting for age and HCV RNA titer indicated that the ISDR type was the most potent predictive factor. CONCLUSION: HCV RNA ISDR type is an important factor for predicting efficacy of IFN-α and ribavirin combination therapy in Korean patients.

The transient receptor potential channel TRPV5 is localized to the apical membrane of the distal renal tubule and plays an important role in the regulation of transepithelial Ca(2+) reabsorption in kidney. We have previously reported that extracellular protons inhibit TRPV5 by binding to glutamate-522 (E522) in the extracellular domain of the channel. We suggested that E522 is an extracellular "pH sensor" and its titration by extracellular protons inhibits TRPV5 via conformational change(s) of the pore helix. We now report that mutation of a pore helix residue glutamate-535 to glutamine (E535Q) enhances the sensitivity of the channel to inhibition by extracellular protons (i.e., shifting the apparent pKa for inhibition by extracellular protons to the more alkaline extracellular pH). The enhancement of extracellular proton-mediated inhibition of E535Q mutant is also dependent on E522. We have also reported that intracellular acidification enhances the sensitivity of TRPV5 to inhibition by extracellular protons. We now find that modulation of the extracellular proton-mediated inhibition by intracellular acidification is preserved in the E535Q mutant. These results provide further support for the idea that pore helix is involved in the regulation of TRPV5 by extracellular protons. Inhibition of TRPV5 by extracellular protons may contribute to hypercalciuria in diseases associated with high acid load.

Mutations of the transmembrane conductance regulator (CFTR) gene in cystic fibrosis lead to dysfunction of the lung, pancreas, and sweat glands, etc. To investigate the possibility of the relationship between lung cancer and the mutations of CFTR gene, we determined amino acid sequences using reverse transcription-polymerase chain reaction (RT-PCR) and DNA sequencing. In this study, the deletion mutation of 508th amino acid in one of nine lung caner patients was found confirming that CFTR gene mutation exists in a Korean lung cancer patient.

Corrigendum: Human zinc fingers as building blocks in the construction of artificial transcription factors