bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular imaging at 1-femtosecond resolution
Authors: Ruan Chong-Yu
Published: October 2016

Loss of a hydrogen atom from acetylene is observed with electron diffraction
Hitting Ebola, to the power of two
Authors: Labrijn Aran F. , Parren Paul W. H. I.
Published: October 2016

Bispecific antibodies are engineered to thwart ebolavirus entry into cells
A short presidential reading list
Authors: Berg Jeremy
Published: October 2016

A new U.S. president will be sworn into office in less than 3 months. Because scientific issues cut across many aspects of modern life, in both the public and private sectors, the president has several challenges. He or she must ensure that the government has access to robust advice about scientific issues to guide policy development. The president must select, and the U.S. Senate must confirm, the leaders for agencies with a substantial science focus. Finally, the new president must set the tone regarding the importance of science in the nation's progress. Two short, 70-year-old documents outline many central issues that are still relevant today.
Clinical Laboratory Testing in the Era of Directly Acting Antiviral Therapies for Hepatitis C
Authors: Wilson Eleanor M. , Rosenthal Elana S. , Kattakuzhy Sarah , Tang Lydia , Kottilil Shyam
Published: October 2016

Directly acting antiviral (DAA) combination therapies for chronic hepatitis C virus (HCV) infection are highly effective, but treatment decisions remain complex. Laboratory testing is important to evaluate a range of viral, host, and pharmacological factors when considering HCV treatment, and patients must be monitored during and after therapy for safety and to assess the viral response. In this review, we discuss the laboratory tests relevant for the treatment of HCV infection in the era of DAA therapy, grouped according to viral and host factors.
Cost Analysis of Implementing Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Plus Real-Time Antimicrobial Stewardship Intervention for Bloodstream Infections
Authors: Patel Twisha S. , Kaakeh Rola , Nagel Jerod L. , Newton Duane W. , Stevenson James G.
Published: October 2016

Studies evaluating rapid diagnostic testing plus stewardship intervention have consistently demonstrated improved clinical outcomes for patients with bloodstream infections. However, the cost of implementing new rapid diagnostic testing can be significant, and such testing usually does not generate additional revenue. There are minimal data evaluating the impact of adding matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for rapid organism identification and dedicating pharmacy stewardship personnel time on the total hospital costs. A cost analysis was performed utilizing patient data generated from the hospital cost accounting system and included additional costs of MALDI-TOF equipment, supplies and personnel, and dedicated pharmacist time for blood culture review and of making interventions to antimicrobial therapy. The cost analysis was performed from a hospital perspective for 3-month blocks before and after implementation of MALDI-TOF plus stewardship intervention. A total of 480 patients with bloodstream infections were included in the analysis: 247 in the preintervention group and 233 in the intervention group. Thirty-day mortality was significantly improved in the intervention group (12% versus 21%,
P
< 0.01), and the mean length of stay was reduced, although the difference was not statistically significant (13.0 ± 16.5 days versus 14.2 ± 16.7 days,
P
= 0.44). The total hospital cost per bloodstream infection was lower in the intervention group ($42,580 versus $45,019). Intensive care unit cost per bloodstream infection accounted for the largest share of the total costs in each group and was also lower in the intervention group ($10,833 versus $13,727). Implementing MALDI-TOF plus stewardship review and intervention decreased mortality for patients with bloodstream infections. Despite the additional costs of implementing MALDI-TOF and of dedicating pharmacy stewardship personnel time to interventions, the total hospital costs decreased by $2,439 per bloodstream infection, for an approximate annual cost savings of $2.34 million.
Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients
Authors: Loeffler Juergen , Hafner Julia , Mengoli Carlo , Wirth Clemens , Heussel Claus Peter , Löffler Claudia , White P. Lewis , Ullmann Andrew J. , Michel Denise , Wiegering Verena , Wölfl Matthias , Schlegel Paul Gerhardt , Einsele Hermann , Springer Jan , Eyrich Matthias
Published: October 2016

Combined biomarker screening is increasingly used to diagnose invasive aspergillosis (IA) in high-risk patients. In adults, the combination of galactomannan (GM) and fungal DNA detection has proven to be beneficial in the diagnosis of IA. Data in purely pediatric cohorts are scarce. Here, we monitored 39 children shortly before and after allogeneic stem cell transplantation twice weekly by use of a commercial GM enzyme-linked immunosorbent assay (ELISA) and a PCR assay based on amplification of the pan-
Aspergillus
ITS1/5.8S ribosomal operon. In addition, clinical data were recorded and classification of IA was performed according to the European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria. Among the 39 high-risk children, we identified 4 patients (10.3%) with probable and 2 (5.1%) with possible IA. All patients with probable IA were repeatedly positive for both tests (means of 9.5 and 6.8 positive GM and PCR samples, respectively), whereas both possible IA cases were detected by PCR. The sensitivity and specificity were, respectively, 67% and 89% for GM and 100% and 63% for PCR. Positive and negative predictive values were, respectively, 50% and 100% for GM and 27% and 100% for PCR. For the combined testing approach, both values were 100%. The number of positive samples seemed to be lower in patients undergoing antifungal therapy. Sporadically positive tests occurred in 12% (GM) and 42% (PCR) of unclassified patients. In summary, our data show that combined monitoring for GM and fungal DNA also results in a high diagnostic accuracy in pediatric patients. Future studies have to determine whether combined testing is suitable for early detection of subclinical disease and how antifungal prophylaxis impacts assay performance.
Influenza A Viruses of Swine (IAV-S) in Vietnam from 2010 to 2015: Multiple Introductions of A(H1N1)pdm09 Viruses into the Pig Population and Diversifying Genetic Constellations of Enzootic IAV-S
Authors: Takemae Nobuhiro , Harada Michiyo , Nguyen Phuong Thanh , Nguyen Tung , Nguyen Tien Ngoc , To Thanh Long , Nguyen Tho Dang , Pham Vu Phong , Le Vu Tri , Do Hoa Thi , Vo Hung Van , Le Quang Vinh Tin , Tran Tan Minh , Nguyen Thanh Duy , Thai Phuong Duy , Nguyen Dang Hoang , Le Anh Quynh Thi , Nguyen Diep Thi , Uchida Yuko , Saito Takehiko
Published: October 2016

Active surveillance of influenza A viruses of swine (IAV-S) involving 262 farms and 10 slaughterhouses in seven provinces in northern and southern Vietnam from 2010 to 2015 yielded 388 isolates from 32 farms; these viruses were classified into H1N1, H1N2, and H3N2 subtypes. Whole-genome sequencing followed by phylogenetic analysis revealed that the isolates represented 15 genotypes, according to the genetic constellation of the eight segments. All of the H1N1 viruses were entirely A(H1N1)pdm09 viruses, whereas all of the H1N2 and H3N2 viruses were reassortants among 5 distinct ancestral viruses: H1 and H3 triple-reassortant (TR) IAV-S that originated from North American pre-2009 human seasonal H1, human seasonal H3N2, and A(H1N1)pdm09 viruses. Notably, 93% of the reassortant IAV-S retained M genes that were derived from A(H1N1)pdm09, suggesting some advantage in terms of their host adaptation. Bayesian Markov chain Monte Carlo analysis revealed that multiple introductions of A(H1N1)pdm09 and TR IAV-S into the Vietnamese pig population have driven the genetic diversity of currently circulating Vietnamese IAV-S. In addition, our results indicate that a reassortant IAV-S with human-like H3 and N2 genes and an A(H1N1)pdm09 origin M gene likely caused a human case in Ho Chi Minh City in 2010. Our current findings indicate that human-to-pig transmission as well as cocirculation of different IAV-S have contributed to diversifying the gene constellations of IAV-S in Vietnam.

IMPORTANCE
This comprehensive genetic characterization of 388 influenza A viruses of swine (IAV-S) isolated through active surveillance of Vietnamese pig farms from 2010 through 2015 provides molecular epidemiological insight into the genetic diversification of IAV-S in Vietnam after the emergence of A(H1N1)pdm09 viruses. Multiple reassortments among A(H1N1)pdm09 viruses and enzootic IAV-S yielded 14 genotypes, 9 of which carried novel gene combinations. The reassortants that carried M genes derived from A(H1N1)pdm09 viruses became predominant, replacing those of the IAV-S that had been endemic in Vietnam since 2011. Notably, one of the novel reassortants likely caused a human case in Vietnam. Given that Vietnam is the second-largest pig-producing country in Asia, continued monitoring of IAV-S is highly important from the viewpoints of both the swine industry and human public health.
An
In Vitro
RNA Synthesis Assay for Rabies Virus Defines Ribonucleoprotein Interactions Critical for Polymerase Activity
Authors: Morin Benjamin , Liang Bo , Gardner Erica , Ross Robin A. , Whelan Sean P. J.
Published: October 2016

We report an
in vitro
RNA synthesis assay for the RNA-dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in
Escherichia coli
and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19 nucleotides (nt) of the rabies virus genome, we demonstrate that L alone initiates synthesis on naked RNA and that P serves to enhance the initiation and processivity of the RdRP. The L-P complex lacks full processivity, which we interpret to reflect the lack of the viral nucleocapsid protein (N) on the template. Using this assay, we define the requirements in P for stimulation of RdRP activity as residues 11 to 50 of P and formally demonstrate that ribavirin triphosphate (RTP) inhibits the RdRP. By comparing the properties of RABV RdRP with those of the related rhabdovirus, vesicular stomatitis virus (VSV), we demonstrate that both polymerases can copy the heterologous promoter sequence. The requirements for engagement of the N-RNA template of VSV by its polymerase are provided by the C-terminal domain (CTD) of P. A chimeric RABV P protein in which the oligomerization domain (OD) and the CTD were replaced by those of VSV P stimulated RABV RdRP activity on naked RNA but was insufficient to permit initiation on the VSV N-RNA template. This result implies that interactions between L and the template N are also required for initiation of RNA synthesis, extending our knowledge of ribonucleoprotein interactions that are critical for gene expression.

IMPORTANCE
The current understanding of the structural and functional significance of the components of the rabies virus replication machinery is incomplete. Although structures are available for the nucleocapsid protein in complex with RNA, and also for portions of P, information on both the structure and function of the L protein is lacking. This study reports the expression and purification of the full-length L protein of RABV and the characterization of its RdRP activity
in vitro
. The study provides a new assay that has utility for screening inhibitors and understanding their mechanisms of action, as well as defining new interactions that are required for RdRP activity.
Pivotal Role of Receptor-Interacting Protein Kinase 1 and Mixed Lineage Kinase Domain-Like in Neuronal Cell Death Induced by the Human Neuroinvasive Coronavirus OC43
Authors: Meessen-Pinard Mathieu , Le Coupanec Alain , Desforges Marc , Talbot Pierre J.
Published: October 2016

Human coronaviruses (HCoV) are respiratory pathogens with neuroinvasive, neurotropic, and neurovirulent properties, highlighting the importance of studying the potential implication of these viruses in neurological diseases. The OC43 strain (HCoV-OC43) was reported to induce neuronal cell death, which may participate in neuropathogenesis. Here, we show that HCoV-OC43 harboring two point mutations in the spike glycoprotein (rOC/U
s183–241
) was more neurovirulent than the wild-type HCoV-OC43 (rOC/ATCC) in mice and induced more cell death in murine and human neuronal cells. To evaluate the role of regulated cell death (RCD) in HCoV-OC43-mediated neural pathogenesis, we determined if knockdown of Bax, a key regulator of apoptosis, or RIP1, a key regulator of necroptosis, altered the percentage of neuronal cell death following HCoV-OC43 infection. We found that Bax-dependent apoptosis did not play a significant role in RCD following infection, as inhibition of Bax expression mediated by RNA interference did not confer cellular protection against the cell death process. On the other hand, we demonstrated that RIP1 and MLKL were involved in neuronal cell death, as RIP1 knockdown and chemical inhibition of MLKL significantly increased cell survival after infection. Taken together, these results indicate that RIP1 and MLKL contribute to necroptotic cell death after HCoV-OC43 infection to limit viral replication. However, this RCD could lead to neuronal loss in the mouse CNS and accentuate the neuroinflammation process, reflecting the severity of neuropathogenesis.

IMPORTANCE
Because they are naturally neuroinvasive and neurotropic, human coronaviruses are suspected to participate in the development of neurological diseases. Given that the strain OC43 is neurovirulent in mice and induces neuronal cell death, we explored the neuronal response to infection by characterizing the activation of RCD. Our results revealed that classical apoptosis associated with the Bax protein does not play a significant role in HCoV-OC43-induced neuronal cell death and that RIP1 and MLKL, two cellular proteins usually associated with necroptosis (an RCD back-up system when apoptosis is not adequately induced), both play a pivotal role in the process. As necroptosis disrupts cellular membranes and allows the release of damage-associated molecular patterns (DAMP) and possibly induces the production of proinflammatory cytokines, it may represent a proinflammatory cell death mechanism that contributes to excessive neuroinflammation and neurodegeneration and eventually to neurological disorders after a coronavirus infection.
The Long and Complicated Relationship between Epstein-Barr Virus and Epithelial Cells
Authors: Hutt-Fletcher Lindsey M.
Published: October 2016

The roles of epithelial cells in infection and persistence of the Epstein-Barr virus (EBV) have long been difficult to resolve. However, recent developments have reinforced the conclusion that these cells are a major site of virus replication and raised the possibility that, like papillomaviruses, EBV has evolved to take advantage of epithelial differentiation to ensure survival, persistence, and spread.
Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase
Authors: Takahata Tatsuro , Takeda Eri , Tobiume Minoru , Tokunaga Kenzo , Yokoyama Masaru , Huang Yu-Lun , Hasegawa Atsuhiko , Shioda Tatsuo , Sato Hironori , Kannagi Mari , Masuda Takao
Published: October 2016

Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in
trans
revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form.

IMPORTANCE
Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.
Myeloid Cell Origins, Differentiation, and Clinical Implications
Authors: Weiskopf Kipp , Schnorr Peter J. , Pang Wendy W. , Chao Mark P. , Chhabra Akanksha , Seita Jun , Feng Mingye , Weissman Irving L.
Published: October 2016

The hematopoietic stem cell (HSC) is a multipotent stem cell that resides in the bone marrow and has the ability to form all of the cells of the blood and immune system. Since its first purification in 1988, additional studies have refined the phenotype and functionality of HSCs and characterized all of their downstream progeny. The hematopoietic lineage is divided into two main branches: the myeloid and lymphoid arms. The myeloid arm is characterized by the common myeloid progenitor and all of its resulting cell types. The stages of hematopoiesis have been defined in both mice and humans. During embryological development, the earliest hematopoiesis takes place in yolk sac blood islands and then migrates to the fetal liver and hematopoietic organs. Some adult myeloid populations develop directly from yolk sac progenitors without apparent bone marrow intermediates, such as tissue-resident macrophages. Hematopoiesis also changes over time, with a bias of the dominating HSCs toward myeloid development as animals age. Defects in myelopoiesis contribute to many hematologic disorders, and some of these can be overcome with therapies that target the aberrant stage of development. Furthermore, insights into myeloid development have informed us of mechanisms of programmed cell removal. The CD47/SIRPα axis, a myeloid-specific immune checkpoint, limits macrophage removal of HSCs but can be exploited by hematologic and solid malignancies. Therapeutics targeting CD47 represent a new strategy for treating cancer. Overall, an understanding of hematopoiesis and myeloid cell development has implications for regenerative medicine, hematopoietic cell transplantation, malignancy, and many other diseases.
Tuberculosis Diagnostics: State of the Art and Future Directions
Authors: Pai Madhukar , Nicol Mark P. , Boehme Catharina C.
Published: October 2016

Rapid and accurate diagnosis is critical for timely initiation of anti-tuberculosis (TB) treatment, but many people with TB (or TB symptoms) do not have access to adequate initial diagnosis. In many countries, TB diagnosis is still reliant on sputum microscopy, a test with known limitations. However, new diagnostics are starting to change the landscape. Stimulated, in part, by the success and rollout of Xpert MTB/RIF, an automated, molecular test, there is now considerable interest in new technologies. The landscape looks promising with a pipeline of new tools, particularly molecular diagnostics, and well over 50 companies actively engaged in product development, and many tests have been reviewed by WHO for policy endorsement. However, new diagnostics are yet to reach scale, and there needs to be greater convergence between diagnostics development and the development of shorter TB drug regimens. Another concern is the relative absence of non-sputum-based diagnostics in the pipeline for children, and of biomarker tests for triage, cure, and latent TB progression. Increased investments are necessary to support biomarker discovery, validation, and translation into clinical tools. While transformative tools are being developed, high-burden countries will need to improve the efficiency of their health care delivery systems, ensure better uptake of new technologies, and achieve greater linkages across the TB and HIV care continuum. While we wait for next-generation technologies, national TB programs must scale up the best diagnostics currently available, and use implementation science to get the maximum impact.
Norovirus Polymerase Fidelity Contributes to Viral Transmission
In Vivo
Authors: Arias Armando , Thorne Lucy , Ghurburrun Elsa , Bailey Dalan , Goodfellow Ian
Published: October 2016

Virus replication fidelity and hence the intrahost genetic diversity of viral populations are known to be intricately linked to viral pathogenesis and tropism as well as to immune and antiviral escape during infection. In this study, we investigated whether changes in replication fidelity can impact the ability of a virus to transmit between susceptible hosts by the use of a mouse model for norovirus. We show that a variant encoding a high-fidelity polymerase is transmitted less efficiently between mice than the wild-type strain. This constitutes the first experimental demonstration that the polymerase fidelity of viruses can impact transmission of infection in their natural hosts. These results provide further insight into potential reasons for the global emergence of pandemic human noroviruses that display alterations in the replication fidelity of their polymerases compared to nonpandemic strains.
Douglas-fir radial growth in interior British Columbia can be linked to long-term oscillations in Pacific and Atlantic sea surface temperatures
Authors: Lo Yueh-Hsin , Blanco Juan A. , Guan Biing T.
Published: October 2016

A major problem in modern dendrochronology is that the methods traditionally used for linking tree-ring growth data to climate records are not well suited to reconstructing low-frequency climatic variations. In this study, we explored the alternative ensemble empirical mode decomposition (EEMD) to detrend tree-ring records and extract climate signals without removing low-frequency information. Tree cores of Pseudotsuga menziesii var. glauca (Mayr.) Franco were examined in a semi-arid forest in southern interior British Columbia, western Canada. Ring width data were decomposed into five oscillatory components (intrinsic mode functions, IMFs) of increasingly longer periodicities. IMF 1 was considered white noise, IMF 2 was used to create the first diameter growth index (DGI-1), and IMF 3 and IMF 4 were combined to create the second diameter growth index (DGI-2), whereas IMF 5 and the residual term together were considered as the trend term. The highest significant cross-correlations between DGI-1 and the NAOAugust, NIÑO12May, and PDOJanuary indices were found at 1-year lags. DGI-2 had positive and persistent correlations with NAOJune and PDOMay at 0- to 3-year lags and with NAOMay at 2- and 3-year lags. Our results indicate that periods of slow growth in the tree-ring record matched periods of drought in the North American Pacific Northwest. Such water-limiting conditions are likely caused by oscillatory patterns in the Pacific Ocean sea surface temperatures that influence precipitation in the Pacific Northwest. These drought events are likely exacerbated by changes in winter precipitation (snowpack) related to oscillations of the Atlantic Ocean sea surface temperatures, highlighting the ecological effects of both oceans on terrestrial ecosystems. Such relationships could not be easily found by traditional tree-ring analyses that remove some of the low-frequency signal, and therefore, we suggest EEMD as an additional tool to establishing tree growth – climate relationships.
LEAST SQUARES MONTE CARLO CREDIT VALUE ADJUSTMENT WITH SMALL AND UNIDIRECTIONAL BIAS
Authors: JOSHI MARK , KWON OH KANG
Published: October 2016

Credit value adjustment (CVA) and related charges have emerged as important risk factors following the Global Financial Crisis. These charges depend on uncertain future values of underlying products, and are usually computed by Monte Carlo simulation. For products that cannot be valued analytically at each simulation step, the standard market practice is to use the regression functions from least squares Monte Carlo method to approximate their values. However, these functions do not necessarily provide accurate approximations to product values over all simulated paths and can result in biases that are difficult to control. Motivated by a novel characterization of the CVA as the value of an option with an early exercise opportunity at a stochastic time, we provide an approximation for CVA and other credit charges that rely only on the sign of the regression functions. The values are determined, instead, by pathwise deflated cash flows. A comparison of CVA for Bermudan swaptions and cancellable swaps shows that the proposed approximation results in much smaller errors than the standard approach of using the regression function values.
Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury
Authors: Jin Younggeon , Blikslager Anthony T.
Published: October 2016

Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions, but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt tight junction barrier function via MLCK activation and myosin light chain (MLC) phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 h followed by reoxygenation in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and FITC-dextran flux. Tight junction proteins and MLCK signaling were assessed by Western blotting, real-time PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) by using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared with control cells, as determined by TER and dextran fluxes ( P < 0.05). The tight junction protein occludin was internalized during A/R injury and relocalized to the region of the tight junction after 4 h of recovery. MLC phosphorylation was significantly increased by A/R injury ( P < 0.05), and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury.
Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury
Authors: Nowak Grazyna , Takacsova-Bakajsova Diana , Megyesi Judit
Published: October 2016

Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia.
The native TRPP2-dependent channel of murine renal primary cilia
Authors: Kleene Steven J. , Kleene Nancy K.
Published: October 2016

Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening monogenic renal disease. ADPKD results from mutations in either of two proteins: polycystin-1 (also known as PC1 or PKD1) or transient receptor potential cation channel, subfamily P, member 2 (TRPP2, also known as polycystin-2, PC2, or PKD2). Each of these proteins is expressed in the primary cilium that extends from many renal epithelial cells. Existing evidence suggests that the cilium can promote renal cystogenesis, while PC1 and TRPP2 counter this cystogenic effect. To better understand the function of TRPP2, we investigated its electrophysiological properties in the native ciliary membrane. We recorded directly from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. In one-third of cilia examined, a large-conductance channel was observed. The channel was not permeable to Cl¯ but conducted cations with permeability ratios PK: PCa: PNaof 1:0.55:0.14. The single-channel conductance ranged from 97 pS in typical physiological solutions to 189 pS in symmetrical 145 mM KCl. Open probability of the channel was very sensitive to membrane depolarization or increasing cytoplasmic free Ca2+in the low micromolar range, with the open probability increasing in either case. Knocking out TRPP2 by CRISPR/Cas9 genome editing eliminated the channel current, establishing it as TRPP2 dependent. Possible mechanisms for activating the TRPP2-dependent channel in the renal primary cilium are discussed.
Intermittent hypobaric hypoxia combined with aerobic exercise improves muscle morphofunctional recovery after eccentric exercise to exhaustion in trained rats
Authors: Rizo-Roca D. , Ríos-Kristjánsson J. G. , Núñez-Espinosa C. , Santos-Alves E. , Gonçalves I. O. , Magalhães J. , Ascensão A. , Pagès T. , Viscor G. , Torrella J. R.
Published: October 2016

Unaccustomed eccentric exercise leads to muscle morphological and functional alterations, including microvasculature damage, the repair of which is modulated by hypoxia. We present the effects of intermittent hypobaric hypoxia and exercise on recovery from eccentric exercise-induced muscle damage (EEIMD). Soleus muscles from trained rats were excised before (CTRL) and 1, 3, 7, and 14 days after a double session of EEIMD protocol. A recovery treatment consisting of one of the following protocols was applied 1 day after the EEIMD: passive normobaric recovery (PNR), a 4-h daily exposure to passive hypobaric hypoxia at 4,000 m (PHR), or hypobaric hypoxia exposure followed by aerobic exercise (AHR). EEIMD produced an increase in the percentage of abnormal fibers compared with CTRL, and it affected the microvasculature by decreasing capillary density (CD, capillaries per mm2) and the capillary-to-fiber ratio (CF). After 14 days, AHR exhibited CD and CF values similar to those of CTRL animals (789 and 3.30 vs. 746 and 3.06) and significantly higher than PNR (575 and 2.62) and PHR (630 and 2.92). Furthermore, VEGF expression showed a significant 43% increase in AHR when compared with PNR. Moreover, after 14 days, the muscle fibers in AHR had a more oxidative phenotype than the other groups, with significantly smaller cross-sectional areas (AHR, 3,745; PNR, 4,502; and PHR, 4,790 µm2), higher citrate synthase activity (AHR, 14.8; PNR, 13.1; and PHR, 12 µmol·min−1·mg−1) and a significant 27% increment in PGC-1α levels compared with PNR. Our data show that hypoxia combined with exercise attenuates or reverses the morphofunctional alterations induced by EEIMD.

NEW & NOTEWORTHY Our study provides new insights into the use of intermittent hypobaric hypoxia combined with exercise as a strategy to recover muscle damage induced by eccentric exercise. We analyzed the effects of hypobaric exposure combined with aerobic exercise on histopathological features of muscle damage, fiber morphofunctionality, capillarization, angiogenesis, and the oxidative capacity of damaged soleus muscle. Most of these parameters were improved after a 2-wk protocol of intermittent hypobaric hypoxia combined with aerobic exercise.
Structural Parameter Identification of Articulated Arm Coordinate Measuring Machines
Authors: Gao Guanbin , Zhang Huaishan , Wu Xing , Guo Yu
Published: October 2016

Precise structural parameter identification of a robotic articulated arm coordinate measuring machine (AACMM) is essential for improving its measuring accuracy, particularly in robotic applications. This paper presents a constructive parameter identification approach for robotic AACMMs. We first develop a mathematical kinematic model of the AACMM based on the Denavit-Hartenberg (DH) approach established for robotic systems. This model is further calibrated and verified via the practical test data. Based on the difference between the calculated coordinates of the AACMM probe via the kinematic model and the given reference coordinates, a parameter identification approach is proposed to estimate the structural parameters in terms of the test data set. The Jacobian matrix is further analyzed to determine the solvability of the identification model. It shows that there are two coupling parameters, which can be removed in the regressor. Finally, a parameter identification algorithm taking the least-square solution of the identification model as the structural parameters by using the obtained poses data is suggested. Practical experiments based on a robotic AACMM test rig are carried out, and the results reveal the effectiveness and robustness of the proposed identification approach.
Endothelium‐Dependent Vasorelaxant Effects of Dealcoholized Wine Powder of Wild Grape (Vitis coignetiae) in the Rat Thoracic Aorta
Authors: Ha Sang Keun , Park Ho-Young , Ryu Mee-Ra , Kim Yoonsook , Park Yongkon
Published: October 2016

The vasorelaxant effects of dealcoholized wild grape (Vitis coignetiae) wine were investigated with isolated rat thoracic aorta. In our present study, we demonstrate that wild grape wine powder (WGWP) induced relaxation of aortic rings preconstricted with norepinephrine in a dose‐dependent manner (at concentrations ranging from 0.1 to 1 mg/mL). The vasorelaxant effect of WGWP was dependent on intact endothelia, which was attenuated by incubation with inhibitors of endothelium‐derived relaxing factors, such as NG‐nitro‐L‐arginine (nitric oxide synthase inhibitor), methylene blue (guanylate cyclase inhibitor), and indomethacin (cyclooxygenase inhibitor). Moreover, treatment with WGWP and atropine (muscarinic receptor antagonist) or diphenylhydramine (histamine receptor antagonist) significantly inhibited endothelium‐dependent vasorelaxation. Our results suggest that WGWP induces relaxation in rat aortic rings in an endothelium‐dependent manner. Results further indicate that this effect occurs via nitric oxide‐cGMP pathway and prostacyclin‐cAMP pathway through a muscarinic receptor and histamine receptor.
The Role ofH. pyloriCagA in Regulating Hormones of Functional Dyspepsia Patients
Authors: Meng Wang-Ping , Wang Zhong-Qiong , Deng Jia-Qi , Liu Yi , Deng Ming-Ming , Lü Mu-Han
Published: October 2016

Helicobacter pylori(H. pylori, Hp) colonizes the stomachs of approximately 20%–80% of humans throughout the world. The Word Healthy Organization (WHO) classifiedH. pylorias a group 1 carcinogenic factor in 1994. Recently, an increasing number of studies has shown an association betweenH. pyloriinfection and various extragastric diseases. Functional dyspepsia (FD) is considered a biopsychosocial disorder with multifactorial pathogenesis, and studies have shown that infection with CagA-positiveH. pyloristrains could explain some of the symptoms of functional dyspepsia. Moreover, CagA-positiveH. pyloristrains have been shown to affect the secretion of several hormones, including 5-HT, ghrelin, dopamine, and gastrin, and altered levels of these hormones might be the cause of the psychological disorders of functional dyspepsia patients. This review describes the mutual effects ofH. pyloriand hormones in functional dyspepsia and provides new insight into the pathogenesis of functional dyspepsia.
Dysregulation of RUNX2/Activin-A Axis upon miR-376c Downregulation Promotes Lymph Node Metastasis in Head and Neck Squamous Cell Carcinoma
Authors: Chang Wei-Min , Lin Yuan-Feng , Su Chia-Yi , Peng Hsuan-Yu , Chang Yu-Chan , Lai Tsung-Ching , Wu Guan-Hsun , Hsu Yuan-Ming , Chi Li-Hsing , Hsiao Jenn-Ren , Chen Chi-Long , Chang Jang-Yang , Shieh Yi-Shing , Hsiao Michael , Shiah Shine-Gwo
Published: October 2016

Epigenetic correlates of the head and neck cancer may illuminate its pathogenic roots. Through a gene set enrichment analysis, we found that the oncogenic transcription factor RUNX2 is widely upregulated in the head and neck squamous cell carcinoma (HNSCC) with lymph node metastasis, where it also predicts poor prognosis in patients with HNSCC. Enforced expression of ectopic RUNX2 promoted the metastatic capabilities of HNSCC, whereas RUNX2 silencing inhibited these features. Mechanistic investigations showed that manipulating levels of activin A (INHBA) could rescue or compromise the RUNX2-mediated metastatic capabilities of HNSCC cells. Furthermore, we found that miR-376c-3p encoded within the 3′-untranslated region of RUNX2 played a pivotal role in regulating RUNX2 expression in highly metastatic HNSCC cells, where it was downregulated commonly. Restoring miR-376c expression in this setting suppressed expression of RUNX2/INHBA axis along with metastatic capability. Clinically, we observed an inverse relationship between miR-376c-3p expression and the RUNX2/INHBA axis in HNSCC specimens. In summary, our results defined a novel pathway in which dysregulation of the RUNX2/INHBA axis due to miR-376c downregulation fosters lymph node metastasis in HNSCC. Cancer Res; 76(24); 7140–50. ©2016 AACR.
Determinants of Light and Intermittent Smoking in the United States: Results from Three Pooled National Health Surveys
Authors: Reyes-Guzman Carolyn M. , Pfeiffer Ruth M. , Lubin Jay , Freedman Neal D. , Cleary Sean D. , Levine Paul H. , Caporaso Neil E.
Published: October 2016

Background: Light and/or intermittent smokers have been the fastest growing segment of cigarette smokers in the United States over the past two decades. Defining their behavioral characteristics is a critical public health priority.

Methods: Our sample included 78,229 U.S. adults from three pooled contemporary population-based surveys: the 2012 NHIS, 2012 NSDUH, and 2011–2012 NHANES. We classified current smokers into four categories (light and intermittent [LITS], light-daily, heavier-intermittent, and heavier-daily) and assessed smoking behaviors, illicit drug use, and mental health indicators using weighted analyses.

Results: Analyses associated smoking categories with nicotine dependence, age of smoking initiation, race/ethnicity, and other demographic and behavioral factors. Compared with heavier-daily smokers, smokers who were LITS were most likely to have mild or no nicotine dependence (weighted odds ratio [OR], 16.92; 95% confidence interval [CI], 13.10–21.85), to start smoking cigarettes regularly after age 21 (OR, 3.42; 95% CI, 2.84–4.12), and to be Hispanic (OR, 5.38; 95% CI, 4.38–6.61). Additional significant results were found for other categories of smokers.

Conclusions: Based on pooled data from three large national surveys, light and/or intermittent smokers differed in smoking, drug use, and mental health behaviors from heavier-daily, former, and never smokers. Notable differences by level of smoking frequency and intensity were observed for nicotine dependence, age of smoking initiation, and race/ethnicity.

Impact: Our results may help focus preventive measures and policies for the growing number of light and/or intermittent smokers in the United States because smoking patterns vary by behavioral and socioeconomic factors. Cancer Epidemiol Biomarkers Prev; 26(2); 228–39. ©2016 AACR.
Inhibition of STAT3 with the Generation 2.5 Antisense Oligonucleotide, AZD9150, Decreases Neuroblastoma Tumorigenicity and Increases Chemosensitivity
Authors: Odate Seiichi , Veschi Veronica , Yan Shuang , Lam Norris , Woessner Richard , Thiele Carol J.
Published: October 2016

Purpose: Neuroblastoma is a pediatric tumor of peripheral sympathoadrenal neuroblasts. The long-term event-free survival of children with high-risk neuroblastoma is still poor despite the improvements with current multimodality treatment protocols. Activated JAK/STAT3 pathway plays an important role in many human cancers, suggesting that targeting STAT3 is a promising strategy for treating high-risk neuroblastoma.

Experimental Design: To evaluate the biologic consequences of specific targeting of STAT3 in neuroblastoma, we assessed the effect of tetracycline (Tet)-inducible STAT3 shRNA and the generation 2.5 antisense oligonucleotide AZD9150 which targets STAT3 in three representative neuroblastoma cell line models (AS, NGP, and IMR32).

Results: Our data indicated that Tet-inducible STAT3 shRNA and AZD9150 inhibited endogenous STAT3 and STAT3 target genes. Tet-inducible STAT3 shRNA and AZD9150 decreased cell growth and tumorigenicity. In vivo, STAT3 inhibition by Tet-inducible STAT3 shRNA or AZD9150 alone had little effect on growth of established tumors. However, when treated xenograft tumor cells were reimplanted into mice, there was a significant decrease in secondary tumors in the mice receiving AZD9150-treated tumor cells compared with the mice receiving ntASO-treated tumor cells. This indicates that inhibition of STAT3 decreases the tumor-initiating potential of neuroblastoma cells. Furthermore, inhibition of STAT3 significantly increased neuroblastoma cell sensitivity to cisplatin and decreased tumor growth and increased the survival of tumor-bearing mice in vivo.

Conclusions: Our study supports the development of strategies targeting STAT3 inhibition in combination with conventional chemotherapy for patients with high-risk neuroblastoma. Clin Cancer Res; 23(7); 1771–84. ©2016 AACR.
Integrative Systemic and Local Metabolomics with Impact on Survival in High-Grade Serous Ovarian Cancer
Authors: Bachmayr-Heyda Anna , Aust Stefanie , Auer Katharina , Meier Samuel M. , Schmetterer Klaus G. , Dekan Sabine , Gerner Christopher , Pils Dietmar
Published: October 2016

Purpose: Cancer metabolism is characterized by alterations including aerobic glycolysis, oxidative phosphorylation, and need of fuels and building blocks.

Experimental Design: Targeted metabolomics of preoperative and follow-up sera, ascites, and tumor tissues, RNA sequencing of isolated tumor cells, local and systemic chemokine, and local immune cell infiltration data from up to 65 high-grade serous ovarian cancer patients and 62 healthy controls were correlated to overall survival and integrated in a Systems Medicine manner.

Results: Forty-three mainly (poly)unsaturated glycerophospholipids and four essential amino acids (citrulline) were significantly reduced in patients with short compared with long survival and healthy controls. The glycerophospholipid fingerprint is identical to the fingerprint from isolated (very) low-density lipoproteins (vLDL), indicating that the source of glycerophospholipids consumed by tumors is (v)LDL. A glycerophospholipid-score (HR, 0.46; P = 0.007) and a 100-gene signature (HR, 0.65; P = 0.004) confirmed the independent impact on survival in training (n = 65) and validation (n = 165) cohorts. High concentrations of LDLs and glycerophospholipids were independently predictors for favorable survival. Patients with low glycerophospholipids presented with more systemic inflammation (C-reactive protein and fibrinogen negatively and albumin positively correlated) but less adaptive immune cell tumor infiltration (lower tumor and immune cell PD-L1 expression), less oxygenic respiration and increased triglyceride biosynthesis in tumor cells, and lower histone expressions, correlating with higher numbers of expressed genes and more transcriptional noise, a putative neo-pluripotent tumor cell phenotype.

Conclusions: Low serum phospholipids and essential amino acids are correlated with worse outcome in ovarian cancer, accompanied by a specific tumor cell phenotype. Clin Cancer Res; 23(8); 2081–92. ©2016 AACR.