Παρασκευή, 19 Φεβρουαρίου 2016

Local 'free-style' perforator flaps in head and neck reconstruction: an update and a useful classification.

Local 'free-style' perforator flaps in head and neck reconstruction: an update and a useful classification.

Plast Reconstr Surg. 2016 Feb 11;

Authors: Kokkoli E, Shih HS, Spyropoulou GA, Jeng SF

PMID: 26890505 [PubMed - as supplied by publisher]



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A Huge Ancient Schwannoma of the Epiglottis.

A Huge Ancient Schwannoma of the Epiglottis.

J Craniofac Surg. 2016 Feb 15;

Authors: Lee DH, Kim JH, Yoon TM, Lee JK, Lim SC

Abstract
Ancient schwannoma of the epiglottis is extremely rare. The authors report the first case of a patient with a huge ancient schwannoma of the epiglottis. Clinicians should consider the possibility that ancient schwannoma may originate in the epiglottis mimicking the other more frequently observed lesions.

PMID: 26890461 [PubMed - as supplied by publisher]



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Retrospective data suggests that the higher prevalence of benign paroxysmal positional vertigo in individuals with type 2 diabetes is mediated by hypertension.

Retrospective data suggests that the higher prevalence of benign paroxysmal positional vertigo in individuals with type 2 diabetes is mediated by hypertension.

J Vestib Res. 2016 Jan 28;25(5-6):233-239

Authors: D'Silva LJ, Staecker H, Lin J, Sykes KJ, Phadnis MA, McMahon TM, Connolly D, Sabus CH, Whitney SL, Kluding PM

Abstract
OBJECTIVE: Benign Paroxysmal Positional Vertigo (BPPV) has been linked to comorbidities like diabetes and hypertension. However, the relationship between type 2 diabetes (DM) and BPPV is unclear. The purpose of this retrospective study was to examine the relationship between DM and BPPV in the presence of known contributors like age, gender and hypertension.
METHODS: A retrospective review of the records of 3933 individuals was categorized by the specific vestibular diagnosis and for the presence of type 2 DM and hypertension. As the prevalence of BPPV was higher in people with type 2 DM compared to those without DM, multivariable logistic regressions were used to identify variables predictive of BPPV. The relationship between type 2 DM, hypertension and BPPV was analyzed using mediation analysis.
RESULTS: BPPV was seen in 46% of individuals with type 2 DM, compared to 37% of individuals without DM (p< 0.001). Forty two percent of the association between type 2 DM and BPPV was mediated by hypertension, and supported hypertension as a complete mediator in the relationship between type 2 DM and BPPV.
CONCLUSIONS: Hypertension may provide the mediating pathway by which diabetes affects the vestibular system. Individuals with complaints of dizziness, with comorbidities including hypertension and diabetes, may benefit from a screening for BPPV.

PMID: 26890424 [PubMed - as supplied by publisher]



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Visual perception of upright: Head tilt, visual errors and viewing eye.

Visual perception of upright: Head tilt, visual errors and viewing eye.

J Vestib Res. 2016 Jan 28;25(5-6):201-209

Authors: Kheradmand A, Gonzalez G, Otero-Millan J, Lasker A

Abstract
BACKGROUND: Perception of upright is often assessed by aligning a luminous line to the subjective visual vertical (SVV).
OBJECTIVE: Here we investigated the effects of visual line rotation and viewing eye on SVV responses and whether there was any change with head tilt.
METHODS: SVV was measured using a forced-choice paradigm and by combining the following conditions in 22 healthy subjects: head position (20° left tilt, upright and 20° right tilt), viewing eye (left eye, both eyes and right eye) and direction of visual line rotation (clockwise [CW] and counter clockwise [CCW]).
RESULTS: The accuracy and precision of SVV responses were not different between the viewing eye conditions in all head positions (P> 0.05, Kruskal-Wallis test). The accuracy of SVV responses was % significantly different between the CW and CCW line rotations (p ≈ 0.0001; Kruskal-Wallis test) and SVV was tilted in the same direction as the line rotation. This effect of line rotation was however not consistent across head tilts and was only present in the upright and right tilt head positions. The accuracy of SVV responses showed a higher variability among subjects in the left head tilt position with no significant difference between the CW and CCW line rotations (P> 0.05; post-hoc Dunn's test).
CONCLUSIONS: In spite of the challenges to the estimate of upright with head tilt, normal subjects did remarkably well irrespective of the viewing eye. The physiological significance of the asymmetry in the effect of line rotation between the head tilt positions is unclear but it %may suggest suggests a lateralizing effect of head tilt on the visual perception of upright.

PMID: 26890421 [PubMed - as supplied by publisher]



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Matricellular Protein Periostin Mediates Intestinal Inflammation through the Activation of Nuclear Factor κB Signaling.

Matricellular Protein Periostin Mediates Intestinal Inflammation through the Activation of Nuclear Factor κB Signaling.

PLoS One. 2016;11(2):e0149652

Authors: Koh SJ, Choi Y, Kim BG, Lee KL, Kim DW, Kim JH, Kim JW, Kim JS

Abstract
Periostin is a matricellular protein that interacts with various integrin molecules on the cell surface. Although periostin is expressed in inflamed colonic mucosa, its role in the regulation of intestinal inflammation remains unclear. We investigated the role of periostin in intestinal inflammation using Postn-deficient (Postn-/-) mice. Intestinal epithelial cells (IECs) were transfected by Postn small interfering RNAs. Periostin expression was determined in colon tissue samples from ulcerative colitis (UC) patients. Oral administration of dextran sulfate sodium (DSS) or rectal administration of trinitrobenzene sulfonic acid, induced severe colitis in wild-type mice, but not in Postn-/- mice. Administration of recombinant periostin induced colitis in Postn-/- mice. The periostin neutralizing-antibody ameliorated the severity of colitis in DSS-treated wild-type mice. Silencing of Postn inhibited inteleukin (IL)-8 mRNA expression and NF-κB DNA-binding activity in IECs. Tumor necrosis factor (TNF)-α upregulated mRNA expression of Postn in IECs, and recombinant periostin strongly enhanced IL-8 expression in combination with TNF-α, which was suppressed by an antibody against integrin αv (CD51). Periostin and CD51 were expressed at significantly higher levels in UC patients than in controls. Periostin mediates intestinal inflammation through the activation of NF-κB signaling, which suggests that periostin is a potential therapeutic target for inflammatory bowel disease.

PMID: 26890265 [PubMed - as supplied by publisher]



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Neuronavigated left temporal continuous theta burst stimulation in chronic tinnitus.

Neuronavigated left temporal continuous theta burst stimulation in chronic tinnitus.

Restor Neurol Neurosci. 2016 Jan 30;

Authors: Schecklmann M, Giani A, Tupak S, Langguth B, Raab V, Polak T, Várallyay C, Harnisch W, Herrmann MJ, Fallgatter AJ

Abstract
PURPOSE: Clinical effects of repetitive transcranial magnetic stimulation (rTMS) in chronic tinnitus are moderate. More precise coil localisation strategies, innovative stimulation protocols, and identification of predictors for treatment response were proposed as promising attempts to enhance treatment efficacy. In this pilot study we investigated neuronavigated continuous theta burst TMS (cTBS).
METHODS: Twenty-three patients received neuronavigated cTBS over the left primary auditory cortex in a randomized sham-controlled trial (verum = 12; sham = 11). Treatment response was evaluated with tinnitus questionnaires and numeric rating scales. Immediate change in numeric rating scales during the first session was used as predictor for treatment response.
RESULTS: Tinnitus was significantly reduced after treatment, but there were no superior effects between verum vs. sham treatment. Immediate change in the first treatment session predicted the response to treatment only in the verum group.
CONCLUSIONS: In our study, verum cTBS was not superior to sham which highlights the persistent need for improving non-invasive brain stimulation techniques for the treatment of tinnitus. Future research should focus on the transfer of positive single session effects to daily treatment trials.

PMID: 26890094 [PubMed - as supplied by publisher]



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Mandarin Tone Identification in Cochlear Implant Users Using Exaggerated Pitch Contours.

Mandarin Tone Identification in Cochlear Implant Users Using Exaggerated Pitch Contours.

Otol Neurotol. 2016 Feb 15;

Authors: He A, Deroche ML, Doong J, Jiradejvong P, Limb CJ

Abstract
OBJECTIVE: To determine whether exaggerating the variations in fundamental frequency (F0) contours of Mandarin-based pitch fluctuations could improve tone identification by cochlear implant (CI) users.
METHODS: Twelve normal-hearing (NH) listeners and 11 CI users were tested for their ability to recognize F0 contours modeled after Mandarin tones, in 4- or 5-alternatives forced-choice paradigms. Two types of stimuli were used: computer-generated complex tones and voice recordings. Four contours were tested with voice recordings: flat, rise, fall, and dip. A fifth contour, peak, was added for complex tones. The F0 range of each contour was varied in an adaptive manner. A maximum-likelihood technique was used to fit a psychometric function to the performance data and extract threshold at 70% accuracy.
RESULTS: As F0 range increased, performance in tone identification improved but did not reach 100% for some CI users, suggesting that confusions between contours could always be made even with extremely exaggerated contours. Compared with NH participants, CI users required substantially larger F0 ranges to identify tones, on the order of 9.3 versus 0.4 semitones. CI users achieved better performance for complex tones than for voice recordings, whereas the reverse was true for NH participants. Confusion matrices showed that the "flat" tone was often a default option when the tone contour's F0 range presented was too narrow for participants to respond correctly.
CONCLUSION: These results demonstrate markedly impaired ability for CI users to identify tonal contours, but suggest that the use of exaggerated pitch contours may be helpful for tonal language perception.

PMID: 26890043 [PubMed - as supplied by publisher]



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Novel mutations of PIK3CA gene in head and neck squamous cell carcinoma.

Novel mutations of PIK3CA gene in head and neck squamous cell carcinoma.

Cancer Biomark. 2016 Feb 2;

Authors: Al-Amri AM, Vatte C, Cyrus C, Chathoth S, Hashim TM, Mohamed YS, Ali RA, Alsaid A, Ali AA

Abstract
BACKGROUND: HNSCC is the sixth most common human cancer globally. In Saudi Arabia, HNSCC accounts for seven percent of all newly diagnosed cancer cases. The PIK3CA is one of the most commonly mutated oncogene in human malignancies, including HNSCC.
OBJECTIVE: The objective of this study is to identify mutations in exon 9 and exon 20 of the PIK3CA gene among Saudi HNSCC patients, determine the frequency of these mutations and correlate with clinical and pathological findings.
METHODS: Histopathologically confirmed paraffin embedded HNSCC tumor tissues from 48 patients were obtained. Capillary sequencing method was used to sequence exons 9 and 20 of the PIK3CA gene. Concurrently, the expression analysis of the PIK3CA and PTEN genes were performed using real-time PCR.
RESULTS: Sixty percent of the samples studied were of pharyngeal cancer. A total of seven mutations were identified in exons 9 and 20 of the PIK3CA gene in 14 HNSCC tumor tissue specimens. The seven mutations encompassed one hot spot mutation E542K, a common mutation T1025T and the five novel mutation comprising three missense and two silent mutations. Interestingly, eight out of the 14 samples with a mutation were of patients with pharyngeal cancer.
CONCLUSION: PIK3CA gene plays a crucial role in carcinogenesis in general and HNSCC in particular. The identification of five novel mutations suggest that Saudis may have different frequencies of somatic genetic alterations that may influence HNSCC compared to other populations.

PMID: 26889984 [PubMed - as supplied by publisher]



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Global Sensory Impairment in Older Adults in the United States.

Global Sensory Impairment in Older Adults in the United States.

J Am Geriatr Soc. 2016 Feb;64(2):306-313

Authors: Correia C, Lopez KJ, Wroblewski KE, Huisingh-Scheetz M, Kern DW, Chen RC, Schumm LP, Dale W, McClintock MK, Pinto JM

Abstract
OBJECTIVES: To determine whether there may be a common mechanism resulting in global sensory impairment of the five classical senses (vision, smell, hearing, touch, and taste) in older adults.
DESIGN: Representative, population-based study.
SETTING: National Social Life, Health, and Aging Project.
PARTICIPANTS: Community-dwelling U.S. adults aged 57 to 85.
MEASUREMENTS: The frequency with which impairment co-occurred across the five senses was estimated as an integrated measure of sensory aging. It was hypothesized that multisensory deficits would be common and reflect global sensory impairment that would largely explain the effects of age, sex, and race on sensory dysfunction.
RESULTS: Two-thirds of subjects had two or more sensory deficits, 27% had just one, and 6% had none. Seventy-four percent had impairment in taste, 70% in touch, 22% in smell, 20% in corrected vision, and 18% in corrected hearing. Older adults, men, African Americans, and Hispanics had greater multisensory impairment (all P < .01). Global sensory impairment largely accounted for the effects of age, sex, and race on the likelihood of impairment in each of the five senses.
CONCLUSION: Multisensory impairment is prevalent in older U.S. adults. These data support the concept of a common process that underlies sensory aging across the five senses. Clinicians assessing individuals with a sensory deficit should consider further evaluation for additional co-occurring sensory deficits.

PMID: 26889840 [PubMed - as supplied by publisher]



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Non-melanoma skin cancer of the head and neck: the aid of reflectance confocal microscopy for the accurate diagnosis and management.

Non-melanoma skin cancer of the head and neck: the aid of reflectance confocal microscopy for the accurate diagnosis and management.

G Ital Dermatol Venereol. 2016 Feb 18;

Authors: Ferrari B, Salgarelli AC, Mandel VD, Bellini P, Reggiani C, Farnetani F, Pellacani G, Magnoni C

Abstract
BACKGOUND: Non-melanoma skin cancer (NMSC) represents the most common cutaneous neoplasms of the head and neck. In recent years, novel non-invasive diagnostic tool have been developed, and among these we have the reflectance confocal microscopy (RCM), that offers the evaluation of the skin at real time with cellular resolution. Numerous studies have identified the main confocal features of skin tumours, demonstrating the good correlation of these features with certain dermatoscopic patterns and histologic findings.
METHODS: The aim of this analysis was to provide new insight into the role of RCM in the diagnosis and management of NMSC of the head and neck. Data comes from the most recent literature, taking into account previous essential reported information in this field. The study eligibility criteria were: studies providing update information, focusing on RCM findings in NMSC, without restrictions for age, sex, ethnicity. A search concerning the role of dermoscopy and RCM in the diagnosis of NMSC was performed on Medline. Duplicated studies, single case report and papers with language other than English were excluded from this study.
RESULTS: RCM clues were analysed for NMSC in association with clinical, dermoscopic and histopathologic findings. Moreover, some new findings have been described and possible applications for NMSC of the head and neck have been discussed.
CONCLUSION: RCM allows tissue imaging in-vivo contributing to a more accurate diagnosis of NMSC of the head and neck, sparing time for the patient and costs for the public health system. RCM can also be used for selection of the biopsy site and it is helpful in defining the surgical safety margins to keep during the excision of skin cancers.

PMID: 26889726 [PubMed - as supplied by publisher]



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Opportunity Cost of Surgical Management of Craniomaxillofacial Trauma.

Opportunity Cost of Surgical Management of Craniomaxillofacial Trauma.

Craniomaxillofac Trauma Reconstr. 2016 Mar;9(1):76-81

Authors: Moses H, Powers D, Keeler J, Erdmann D, Marcus J, Puscas L, Woodard C

Abstract
The provision of trauma care is a financial burden, continually associated with low reimbursement, and shifts the economic burden to major trauma centers and providers. Meanwhile, the volume of craniomaxillofacial (CMF) trauma and the number of surgically managed facial fractures are unchanged. Past financial analyses of cost and reimbursement for facial trauma are limited to mandibular and midface injuries, consistently revealing low reimbursement. The incurred financial burden also coincides with the changing landscape of health insurance. The goal of this study is to determine the opportunity cost of operative management of facial trauma at our institution. From our CMF database of greater than 3,000 facial fractures, the physician charges, collections, and relative value units (RVUs) for CMF trauma per year from 2007 to 2013 were compared with a general plastic surgery and otolaryngology population undergoing operative management during this same period. Collection rates were analyzed to assess if a significant difference exists between reimbursement for CMF and non-CMF cases. Results revealed a significant difference between the professional collection rate for operative CMF trauma and that for other operative procedures (17.25 vs. 29.61%, respectively; p < 0.0001). The average number of RVUs billed per provider for CMF trauma declines significantly, from greater than 700 RVUs to 300 over the study period, despite a stable volume. Surgical management of CMF trauma generates an unfavorable financial environment. The large opportunity cost associated with offering this service is a potential threat to the sustainability of providing care for this population.

PMID: 26889352 [PubMed]



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Reconstructive Indications of Anterolateral Thigh Free Flaps in Head and Neck Reconstruction.

Reconstructive Indications of Anterolateral Thigh Free Flaps in Head and Neck Reconstruction.

Craniomaxillofac Trauma Reconstr. 2016 Mar;9(1):40-5

Authors: Khadakban D, Kudpaje A, Thankappan K, Jayaprasad K, Gorasia T, Vidhyadharan S, Mathew J, Sharma M, Iyer S

Abstract
Anterolateral thigh (ALT) free flap is a common flap with multitude of indications. The purpose of this article is to review the reconstructive indications of the flap in head and neck defects. This is a retrospective study of 194 consecutive ALT flaps. Data including patient characteristics (age, sex, comorbidities), disease characteristics (histology, T stage), and flap characteristics (size of the flap, type of closure of ALT donor site) were collected. The outcome in terms of flap success rate, surgical, and donor site morbidity were studied. A total of 194 flaps were performed in 193 patients over a period of 10 years. Mean age of the patients was 55 years (range 16-80 years). Out of the 193 patients, 91 (47.1%) patients had oromandibular defects, 52 (26.9%) had tongue defects, 15 (7.7%) had pharyngeal defects, 17 (8.8%) had skull base defects, 4 (2%) had scalp defects, and 14 (7.2%) had contour defects of the neck. The overall flap success rate was 95.8% (8 total flap loss out of 194). Hypertrophic scar was the commonest donor site problem seen in 20 (10.3%) patients. This study shows the versatility of free ALT flap in head and neck reconstruction. It is a reliable and safe. Donor site morbidity is minimal.

PMID: 26889347 [PubMed]



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Uninsured and Medicaid patients have higher overall and cancer-specific mortality rates - News-Medical.net


OncLive

Uninsured and Medicaid patients have higher overall and cancer-specific mortality rates
News-Medical.net
Compared to patients with non-Medicaid insurance, uninsured patients and patients with Medicaid are more likely to present with advanced stages of head and neck cancer and have higher overall and cancer-specific mortality rates, according to research ...
Chemoradiation Improves OS for Elderly Patients With Head and Neck CancersOncLive
Study links health insurance status and head and neck cancer diagnoses, outcomesEurekAlert (press release)
Uninsured and Medicaid Patients with Head and Neck Cancer More Likely to Present with Advanced Tumors ...Newswise (press release)

all 76 news articles »


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Is Alcohol the New Tobacco? - CounterPunch


CounterPunch

Is Alcohol the New Tobacco?
CounterPunch
According to the National Institute of Health even just regular alcohol consumption “…increases the risk of developing cancers of the mouth, throat, larynx, esophagus, liver, breast, colon and rectum.” The CAMH calculates the cost of alcohol use in ...

and more »


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Paper sensor to track alcohol-related health issues: Indian scientists - Daijiworld.com


Paper sensor to track alcohol-related health issues: Indian scientists
Daijiworld.com
Kolkata, Feb 19 (IANS): Indian scientists have designed a paper sensor that has the potential to track health adversities associated with alcoholism, which they say is a step forward in the realm of low-priced point-of-care (POC) diagnostics in the ...

and more »


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Response to Qian and Colvin: Zinc-mediated Regulation of the Cardiac Ryanodine Receptor Occurs via Multiple Binding Sites [Letters to the Editor]

This is a response to a letter by Qian and Colvin (1).We would like to thank Qian and Colvin for their interest in our recent publication (2) where we demonstrate for the first time that Zn2+ acts as a high affinity activator of the cardiac ryanodine receptor (RyR2). We are aware that BAPTA (2,2′-(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid) is not a Ca2+-specific chelator and can also bind Zn2+ when present. The purpose of the experiment represented in Fig. 4 was to show that Zn2+ can directly activate RyR2 when levels of Ca2+ are subactivating rather than to provide the absolute Zn2+ concentration required for Zn2+-dependent channel openings. This was addressed in the experiments carried out in the absence of BAPTA, where our data reveal that Zn2+ is the primary activating ligand of RyR2 at concentrations >1 nm (Figs. 1 and 3). The estimates of free Zn2+ levels in the presence of BAPTA offered by Qian and Colvin in no way alter the interpretation of our data, and it is unclear why this led them to speculate that the action of Zn2+ is through a single site on the channel (1). A single-site model is not consistent with the finding that 100 pm Zn2+ sensitizes Ca2+-mediated RyR2 activity yet higher concentrations of Zn2+ (1–100 nm) enable switching from Ca2+-dependent to Ca2+-independent gating. Thus, separate Zn2+ sites must exist to enable Ca2+ sensitization and Zn2+ activation, respectively. A single-site model is also not consistent with the observation that very high concentrations of Zn2+ (1 mm)...

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Zinc Modulation of Cardiac Ryanodine Receptor Gating: Alternate Interpretation of the Interplay between Zinc and Calcium [Letters to the Editor]

Woodier et al. (1) report that the cardiac type 2 ryanodine receptor channel (RyR2) shows two modes (sites) of regulation by zinc. When calcium is activating the channel, zinc increases channel Po around 100 pm (high affinity), whereas in the absence of calcium, zinc must reach 100 nm (low affinity) before channel Po is increased (compare Fig. 1 with Fig. 4, model in Fig. 10). We would like to offer an alternate interpretation of these data. In order to achieve nominally 0 free calcium, 1 mm BAPTA (2,2′-(ethylenedioxy)dianiline-N,N,N′,N′-tetraacetic acid) was added. We believe the primary reason that higher zinc (100 nm) must be added to modulate RyR2 gating in 0 free calcium is that zinc is chelated by BAPTA as well. It is not possible to use BAPTA to selectively chelate calcium without similarly affecting free zinc concentrations because Zn2+ binds to BAPTA (Kd = 7.9 nm) with greater affinity than Ca2+ (Kd = 110 nm) (2–4). Using these values, one can estimate free zinc concentrations under the conditions given in Fig. 4 (with the addition of 1 mm BAPTA). With 1 nm zinc added, the free zinc concentration will be ∼0.1 fm, and with 100 μm zinc added, the free zinc concentration will be ∼1 nm. It is noteworthy that 1 nm free zinc is approximately the same concentration range that modulates gating with calcium present (Fig. 1). Thus, the simplest interpretation of these data is that RyR2 has a single high affinity (1 nm) zinc binding site that...

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TET Kinetics [Gene Regulation]

The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1–3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the Km value of Tets 1 and 2 for O2 is 30 μm, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe2+ and 2-oxoglutarate-binding residues increased the Km values for these factors 30–80-fold and reduced the Vmax values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC50 values ∼400–500 μm. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIFα stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization.

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Novel Glycosaminoglycan Binding Sites on CXCL1/MGSA [Immunology]

In humans, the chemokine CXCL1/MGSA (hCXCL1) plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating the directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We addressed this by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using NMR spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding and that they are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as α-domain) has also been identified previously as the GAG-binding domain for the related chemokine CXCL8/IL-8. The second domain, consisting of residues from the N terminus, 40s turn, and third β-strand (defined as β-domain) is novel. Eliminating β-domain binding by mutagenesis does not perturb α-domain binding, indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that the GAG-bound hCXCL1 completely occlude receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.

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Filamin A and Vpu Anti-tetherin Activity [Cell Biology]

Tetherin, also known as bone marrow stromal antigen 2 (BST-2), inhibits the release of a wide range of enveloped viruses, including human immunodeficiency virus, type 1 (HIV-1) by directly tethering nascent virions to the surface of infected cells. The HIV-1 accessary protein Vpu counteracts tetherin restriction via sequestration, down-regulation, and/or displacement mechanisms to remove tetherin from sites of virus budding. However, the exact mechanism of Vpu-mediated antagonism of tetherin restriction remains to be fully understood. Here we report a novel role for the actin cross-linking regulator filamin A (FLNa) in Vpu anti-tetherin activities. We demonstrate that FLNa associates with tetherin and that FLNa modulates tetherin turnover. FLNa deficiency was found to enhance cell surface and steady-state levels of tetherin expression. In contrast, we observed that overexpression of FLNa reduced tetherin expression levels both on the plasma membrane and in intracellular compartments. Although FLNb shows high amino acid sequence similarity with FLNa, we reveal that only FLNa, but not FLNb, plays an essential role in tetherin turnover. We further showed that FLNa deficiency inhibited Vpu-mediated enhancement of virus release through interfering with the activity of Vpu to down-regulate cellular tetherin. Taken together, our studies suggest that Vpu hijacks the FLNa function in the modulation of tetherin to neutralize the antiviral factor tetherin. These findings may provide novel strategies for the treatment of HIV-1 infection.

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A Domain-swapped Misfolded Conformation in H{gamma}D-crystallin [Protein Structure and Folding]

Cataract is a protein misfolding disease where the size of the aggregate is directly related to the severity of the disorder. However, the molecular mechanisms that trigger the onset of aggregation remain unknown. Here we use a combination of protein engineering techniques and single-molecule force spectroscopy using atomic force microscopy to study the individual unfolding pathways of the human γD-crystallin, a multidomain protein that must remain correctly folded during the entire lifetime to guarantee lens transparency. When stretching individual polyproteins containing two neighboring HγD-crystallin monomers, we captured an anomalous misfolded conformation in which the β1 and β2 strands of the N terminus domain of two adjacent monomers swap. This experimentally elusive domain-swapped conformation is likely to be responsible for the increase in molecular aggregation that we measure in vitro. Our results demonstrate the power of force spectroscopy at capturing rare misfolded conformations with potential implications for the understanding of the molecular onset of protein aggregation.

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Novel Naphthalimide Restores p53 Function [Signal Transduction]

p53 inactivation is a hallmark in non-small-cell lung cancer (NSCLC). It is therefore highly desirable to develop tumor-specific treatment for NSCLC therapy by restoring p53 function. Herein, a novel naphthalimide compound, NA-17, was identified as a promising drug candidate in view of both its anticancer activity and mechanism of action. NA-17 exhibited strong anticancer activity on a broad range of cancer cell lines but showed low toxicity to normal cell lines, such as HL-7702 and WI-38. Moreover, NA-17 showed p53-dependent inhibition selectivity in different NSCLC cell lines due to the activation state of endogenous p53 in the background level. Further studies revealed that NA-17 caused cell cycle arrest at the G1 phase, changed cell size, and induced apoptosis and cell death by increasing the proportion of sub-G1 cells. Molecular mechanism studies suggested that targeted accumulation of phospho-p53 in mitochondria and nuclei induced by NA-17 resulted in activation of Bak and direct binding of phospho-p53 to the target DNA sequences, thereby evoking cell apoptosis and cell cycle arrest and eventually leading to irreversible cancer cell inhibition. This work provided new insights into the molecular interactions and anticancer mechanisms of phospho-p53-dependent naphthalimide compounds.

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Thiol-disulfide Biochemistry of Human TRPV1 [Molecular Biophysics]

Transient receptor potential vanilloid 1 (TRPV1) channel is a tetrameric protein that acts as a sensor for noxious stimuli such as heat and for diverse inflammatory mediators such as oxidative stress to mediate nociception in a subset of sensory neurons. In TRPV1 oxidation sensing, cysteine (Cys) oxidation has been considered as the principle mechanism; however, its biochemical basis remains elusive. Here, we characterize the oxidative status of Cys residues in differential redox environments and propose a model of TRPV1 activation by oxidation. Through employing a combination of non-reducing SDS-PAGE, electrophysiology, and mass spectrometry we have identified the formation of subunit dimers carrying a stable intersubunit disulfide bond between Cys-258 and Cys-742 of human TRPV1 (hTRPV1). C258S and C742S hTRPV1 mutants have a decreased protein half-life, reflecting the role of the intersubunit disulfide bond in supporting channel stability. Interestingly, the C258S hTRPV1 mutant shows an abolished response to oxidants. Mass spectrometric analysis of Cys residues of hTRPV1 treated with hydrogen peroxide shows that Cys-258 is highly sensitive to oxidation. Our results suggest that Cys-258 residues are heterogeneously modified in the hTRPV1 tetrameric complex and comprise Cys-258 with free thiol for oxidation sensing and Cys-258, which is involved in the disulfide bond for assisting subunit dimerization. Thus, the hTRPV1 channel has a heterogeneous subunit composition in terms of both redox status and function.

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PRIP Deficiency Enhances Energy Expenditure [Cell Biology]

Phospholipase C-related catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate-binding protein, and was later found to be involved in a variety of cellular events, particularly those related to protein phosphatases. We previously reported that Prip knock-out (KO) mice exhibit a lean phenotype with a small amount of white adipose tissue. In the present study, we examined whether PRIP is involved in energy metabolism, which could explain the lean phenotype, using high-fat diet (HFD)-fed mice. Prip-KO mice showed resistance to HFD-induced obesity, resulting in protection from glucose metabolism dysfunction and insulin resistance. Energy expenditure and body temperature at night were significantly higher in Prip-KO mice than in wild-type mice. Gene and protein expression of uncoupling protein 1 (UCP1), a thermogenic protein, was up-regulated in Prip-KO brown adipocytes in thermoneutral or cold environments. These phenotypes were caused by the promotion of lipolysis in Prip-KO brown adipocytes, which is triggered by up-regulation of phosphorylation of the lipolysis-related proteins hormone-sensitive lipase and perilipin, followed by activation of UCP1 and/or up-regulation of thermogenesis-related genes (e.g. peroxisome proliferator-activated receptor-γ coactivator-1α). The results indicate that PRIP negatively regulates UCP1-mediated thermogenesis in brown adipocytes.

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Purification of the Active Mitochondrial Supercomplex [Bioenergetics]

To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8–35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.

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Nuclear {beta}-Catenin Activation in Colitis [Signal Transduction]

Wnt/β-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser-552 phosphorylated β-catenin (pβ-CatSer-552) in intestinal epithelial cells (IEC) during colitis and colitis-associated cancer. Data here delineate a novel multiprotein cytosolic complex (MCC) involved in β-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with β-catenin, 14-3-3ζ, Akt, and p110α. MCC levels in IEC increase in colitis and colitis-associated cancer patients. IEC-specific p85α-deficient (p85ΔIEC) mice develop more severe dextran sodium sulfate colitis due to delayed ulcer healing and reduced epithelial β-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces β-catenin signaling. Despite worse colitis, p85ΔIEC mice have reduced tumor burden after azoxymethane/dextran sodium sulfate treatment. Together the data indicate that the β-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.

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{beta}-Adrenergic Regulation of Ito [Signal Transduction]

The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory β-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of β-adrenergic receptors (β-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic β-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic β-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by β-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as β-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic β-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation.

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Conformational Disorder in the Most Immature SOD1 [Molecular Bases of Disease]

Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.

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Adverse Cardiac Remodeling in Myozap-/- Mice [Signal Transduction]

The intercalated disc (ID) is a “hot spot” for heart disease, as several ID proteins have been found mutated in cardiomyopathy. Myozap is a recent addition to the list of ID proteins and has been implicated in serum-response factor signaling. To elucidate the cardiac consequences of targeted deletion of myozap in vivo, we generated myozap-null mutant (Mzp−/−) mice. Although Mzp−/− mice did not exhibit a baseline phenotype, increased biomechanical stress due to pressure overload led to accelerated cardiac hypertrophy, accompanied by “super”-induction of fetal genes, including natriuretic peptides A and B (Nppa/Nppb). Moreover, Mzp−/− mice manifested a severe reduction of contractile function, signs of heart failure, and increased mortality. Expression of other ID proteins like N-cadherin, desmoplakin, connexin-43, and ZO-1 was significantly perturbed upon pressure overload, underscored by disorganization of the IDs in Mzp−/− mice. Exploration of the molecular causes of enhanced cardiac hypertrophy revealed significant activation of β-catenin/GSK-3β signaling, whereas MAPK and MKL1/serum-response factor pathways were inhibited. In summary, myozap is required for proper adaptation to increased biomechanical stress. In broader terms, our data imply an essential function of the ID in cardiac remodeling beyond a mere structural role and emphasize the need for a better understanding of this molecular structure in the context of heart disease.

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Red-shifted Chloride Channelrhodopsin [Membrane Biology]

Chloride conducting channelrhodopsins (ChloCs) are new members of the optogenetic toolbox that enable neuronal inhibition in target cells. Originally, ChloCs have been engineered from cation conducting channelrhodopsins (ChRs), and later identified in a cryptophyte alga genome. We noticed that the sequence of a previously described Proteomonas sulcata ChR (PsChR1) was highly homologous to the naturally occurring and previously reported ChloCs GtACR1/2, but was not recognized as an anion conducting channel. Based on electrophysiological measurements obtained under various ionic conditions, we concluded that the PsChR1 photocurrent at physiological conditions is strongly inward rectifying and predominantly carried by chloride. The maximum activation was noted at excitation with light of 540 nm. An initial spectroscopic characterization of purified protein revealed that the photocycle and the transport mechanism of PsChR1 differ significantly from cation conducting ChRs. Hence, we concluded that PsChR1 is an anion conducting ChR, now renamed PsACR1, with a red-shifted absorption suited for multicolor optogenetic experiments in combination with blue light absorbing cation conducting ChRs.

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Icsbp Terminates Emergency Granulopoiesis [Molecular Bases of Disease]

Emergency granulopoiesis occurs in response to infectious or inflammatory challenge and is a component of the innate immune response. Some molecular events involved in initiating emergency granulopoiesis are known, but termination of this process is less well defined. In this study, we found that the interferon consensus sequence binding protein (Icsbp/Irf8) was required to terminate emergency granulopoiesis. Icsbp is an interferon regulatory transcription factor with leukemia suppressor activity. Expression of Icsbp is decreased in chronic myeloid leukemia, and Icsbp−/− mice exhibit progressive granulocytosis with evolution to blast crisis, similar to the course of human chronic myeloid leukemia. In this study, we found aberrantly sustained granulocyte production in Icsbp−/− mice after stimulation of an emergency granulopoiesis response. Icsbp represses transcription of the genes encoding Fas-associated phosphatase 1 (Fap1) and growth arrest-specific 2 (Gas2) and activates genes encoding Fanconi C and F. After stimulation of emergency granulopoiesis, we found increased and sustained expression of Fap1 and Gas2 in bone marrow myeloid progenitor cells from Icsbp−/− mice in comparison with the wild type. This was associated with resistance to Fas-induced apoptosis and increased β-catenin activity in these cells. We also found that repeated episodes of emergency granulopoiesis accelerated progression to acute myeloid leukemia in Icsbp−/− mice. This was associated with impaired Fanconi C and F expression and increased sensitivity to DNA damage in bone marrow myeloid progenitors. Our results suggest that impaired Icsbp expression enhances leukemogenesis by deregulating processes that normally limit granulocyte expansion during the innate immune response.

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Hyaluronan in Fibroblast-Cancer Cell Interaction [Glycobiology and Extracellular Matrices]

The aim of this study was to characterize the interaction of KYSE-410, an esophageal squamous cell carcinoma cell line, and fibroblasts with respect to the extracellular matrix component hyaluronan (HA) and chemokine expression. KYSE-410 cells induced the mRNA expression of HA synthase 2 (Has2) in normal skin fibroblasts (SF) only in direct co-cultures. Parallel to Has2 mRNA, Has2 antisense RNA (Has2os2) was up-regulated in co-cultures. Knockdown of LEF1, a downstream target of Wnt signaling, abrogated Has2 and Has2os2 induction. After knockdown of Has2 in SF, significantly less α-smooth muscle actin expression was detected in co-cultures. Moreover, it was investigated whether the phenotype of KYSE-410 was affected in co-culture with SF and whether Has2 knockdown in SF had an impact on KYSE-410 cells in co-culture. However, no effects on epithelial-mesenchymal transition markers, proliferation, and migration were detected. In addition to Has2 mRNA, the chemokine CCL5 was up-regulated and CCL11 was down-regulated in SF in co-culture. Furthermore, co-cultures of KYSE-410 cells and cancer-associated fibroblasts (CAF) were investigated. Similar to SF, Has2 and Ccl5 were up-regulated and Ccl11 was down-regulated in CAF in co-culture. Importantly and in contrast to SF, inhibiting HA synthesis by 4-methylumbelliferone abrogated the effect of co-culture on Ccl5 in CAF. Moreover, HA was found to promote adhesion of CD4+ but not CD8+ cells to xenogaft tumor tissues. In conclusion, direct co-culture of esophageal squamous cell carcinoma and fibroblasts induced stromal HA synthesis via Wnt/LEF1 and altered the chemokine profile of stromal fibroblasts, which in turn may affect the tumor immune response.

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