Enhanced and controlled chromatin extraction from FFPE tissues and the application to ChIP-seq.
Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays. During rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65 °C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with targeted ChIP-qPCR and genome-wide ChIP-seq approaches. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37 °C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45-60 °C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65 °C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays. This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin. This approach may provide a way to circumvent the over-fixed nature of FFPE tissues for future technology development.


Antisocial symptoms decrease to normal levels in long-term abstinence.
We have previously shown highly elevated antisocial symptoms and measures of social deviance proneness and antisocial disposition in long-term abstinent alcohol dependence versus non-substance-abusing controls (NSAC). Current antisocial symptoms were reduced to subdiagnostic levels in long-term abstinence; however, the number of current symptoms was not measured beyond its being subdiagnostic. Here we measured social deviance proneness, antisocial disposition, and both lifetime and current antisocial symptoms in short-term and long-term abstinent substance-dependent and NSAC samples. Lifetime antisocial symptoms (and diagnoses) and social deviance proneness and antisocial disposition were highly elevated in both short- and long-term abstinence, replicating earlier findings. Current antisocial symptoms were dramatically reduced in long-term versus short-term abstinent samples, close to levels in controls. In contrast, social deviance proneness and antisocial disposition remain highly elevated in long-term abstinence. These findings suggest that antisocial behavior is reduced in extended abstinence, despite continued elevated social deviance proneness an antisocial disposition. This suggests a top-down model in extended abstinence, whereby executive control inhibits deviance-prone tendencies.


PD-1 gene polymorphism in children with subacute sclerosing panencephalitis.
Subacute sclerosing panencephalitis (SSPE) is a progressive inflammatory and degenerative disorder of the central nervous system. Several factors influence the risk of chronic brain infection with the mutant measles virus. However, to date, no pathogenic mechanism that may predispose to SSPE has been determined. Studies have indicated that specific polymorphisms in certain host genes are probably involved in impairing the ability of host immune cells to eradicate the measles virus in SSPE patients. Programmed cell death protein 1 (PD-1), a member of the CD28 family, is a negative regulator of the immune system. The purpose of our study was to investigate whether PD-1 gene polymorphisms affect susceptibility to the development of SSPE in Turkish children. In total, 109 subjects (54 SSPE patients and 55 healthy controls) were genotyped for the PD-1.9 C/T (rs2227982) single-nucleotide polymorphism (SNP). The distributions of T alleles in the PD-1.9 polymorphism in SSPE patients and healthy controls were 2.8 and 10.9%, respectively. There was a statistically significant difference between the groups; the 95% confidence interval (CI) was 0.06 to 0.85 and the odds ratio (OR) was 0.23 (χ(2) test). Thus, we identified an association between SSPE and the PD-1 rs2227982 gene polymorphism; the frequency of T alleles was higher in controls than in SSPE patients.


Superficial malignant peripheral nerve sheath tumor: a rare and challenging diagnosis.
We reviewed the clinicopathologic features of 5 cases of malignant peripheral nerve sheath tumor (MPNST) manifesting in superficial locations associated with cutaneous neurofibromas (4 cases) or superficial peripheral nerve (1 case). Four cases had spindle cell morphologic features and were at least focally positive for S-100 protein, whereas the associated benign neural elements had more extensive S-100 immunoreactivity. The single epithelioid case was diffusely and strongly positive for S-100 protein. Melanoma markers, epithelial membrane antigen, glial fibrillary acidic protein, neurofilament, pancytokeratin (AE1/AE3), CD34, smooth muscle actin, and desmin were negative in all cases. There were no local recurrences, but 3 patients died of metastatic disease within 2 to 30 months (median, 21 months). MPNSTs can occur in a superficial location and may have an aggressive clinical course. Immunohistochemical markers are helpful in excluding other lesions in the differential diagnosis. However, identification of a benign precursor or origin from a nerve may be the most definitive way to properly classify these rare lesions.


Genome-wide analysis of the RpoN regulon in Geobacter sulfurreducens.
The role of the RNA polymerase sigma factor RpoN in regulation of gene expression in Geobacter sulfurreducens was investigated to better understand transcriptional regulatory networks as part of an effort to develop regulatory modules for genome-scale in silico models, which can predict the physiological responses of Geobacter species during groundwater bioremediation or electricity production. An rpoN deletion mutant could not be obtained under all conditions tested. In order to investigate the regulon of the G. sulfurreducens RpoN, an RpoN over-expression strain was made in which an extra copy of the rpoN gene was under the control of a taclac promoter. Combining both the microarray transcriptome analysis and the computational prediction revealed that the G. sulfurreducens RpoN controls genes involved in a wide range of cellular functions. Most importantly, RpoN controls the expression of the dcuB gene encoding the fumarate/succinate exchanger, which is essential for cell growth with fumarate as the terminal electron acceptor in G. sulfurreducens. RpoN also controls genes, which encode enzymes for both pathways of ammonia assimilation that is predicted to be essential under all growth conditions in G. sulfurreducens. Other genes that were identified as part of the RpoN regulon using either the computational prediction or the microarray transcriptome analysis included genes involved in flagella biosynthesis, pili biosynthesis and genes involved in central metabolism enzymes and cytochromes involved in extracellular electron transfer to Fe(III), which are known to be important for growth in subsurface environment or electricity production in microbial fuel cells. The consensus sequence for the predicted RpoN-regulated promoter elements is TTGGCACGGTTTTTGCT. The G. sulfurreducens RpoN is an essential sigma factor and a global regulator involved in a complex transcriptional network controlling a variety of cellular processes.


Functional imaging for regenerative medicine.
In vivo imaging is a platform technology with the power to put function in its natural structural context. With the drive to translate stem cell therapies into pre-clinical and clinical trials, early selection of the right imaging techniques is paramount to success. There are many instances in regenerative medicine where the biological, biochemical, and biomechanical mechanisms behind the proposed function of stem cell therapies can be elucidated by appropriate imaging. Imaging techniques can be divided according to whether labels are used and as to whether the imaging can be done in vivo. In vivo human imaging places additional restrictions on the imaging tools that can be used. Microscopies and nanoscopies, especially those requiring fluorescent markers, have made an extraordinary impact on discovery at the molecular and cellular level, but due to their very limited ability to focus in the scattering tissues encountered for in vivo applications they are largely confined to superficial imaging applications in research laboratories. Nanoscopy, which has tremendous benefits in resolution, is limited to the near-field (e.g. near-field scanning optical microscope (NSNOM)) or to very high light intensity (e.g. stimulated emission depletion (STED)) or to slow stochastic events (photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)). In all cases, nanoscopy is limited to very superficial applications. Imaging depth may be increased using multiphoton or coherence gating tricks. Scattering dominates the limitation on imaging depth in most tissues and this can be mitigated by the application of optical clearing techniques that can impose mild (e.g. topical application of glycerol) or severe (e.g. CLARITY) changes to the tissue to be imaged. Progression of therapies through to clinical trials requires some thought as to the imaging and sensing modalities that should be used. Smoother progression is facilitated by the use of comparable imaging modalities throughout the discovery and trial phases, giving label-free techniques an advantage wherever they can be used, although this is seldom considered in the early stages. In this paper, we will explore the techniques that have found success in aiding discovery in stem cell therapies and try to predict the likely technologies best suited to translation and future directions.


Epidural spinal cord compression from metastatic cancer: clinical features and management.
We retrospectively analyzed thirty-three patients (21 males, 12 females) with malignancy induced spinal cord compression (SCC). The mean age of the patients was 42.8 years and almost half (51%) of them presented with SCC. Mean duration of symptoms was 4.5 months and the mean interval between the original diagnosis of cancer and the development of SCC was 14.6 months. Back pain was the most frequent (97%) symptom with an equal number of patients having subjective or objective evidence of lower limb weakness. Majority (73%) of the patients were non-ambulatory at the time of diagnosis. Spinal level involvement was mostly thoracic (62%) followed by lumber (38%). Breast cancer was the commonest underlying malignancy (21%). Lung (12%), prostrate (12%), multiple myeloma (9%), and carcinoma with unknown primary (12%) were also frequently encountered. There was an overall response rate of 22% to the therapeutic interventions: mostly observed in the ambulatory patients. Only 7% of the non-ambulatory patients regained ability to walk. None of the responders had bladder or bowel dysfunction. Twenty-two percent of the responders are still ambulatory with a mean follow-up of six months.


RIP-seq analysis of eukaryotic Sm proteins identifies three major categories of Sm-containing ribonucleoproteins.
Sm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions. We developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs. This study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism.


Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene.
The in vivo biological function of a steroid receptor coactivator was assessed in mice in which the SRC-1 gene was inactivated by gene targeting. Although in both sexes the homozygous mutants were viable and fertile, target organs such as uterus, prostate, testis, and mammary gland exhibited decreased growth and development in response to steroid hormones. Expression of RNA encoding TIF2, a member of the SRC-1 family, was increased in the SRC-1 null mutant, perhaps compensating partially for the loss of SRC-1 function in target tissues. The results indicate that SRC-1 mediates steroid hormone responses in vivo and that loss of its coactivator function results in partial resistance to hormone.


The Chitranjan S. Ranawat Award : No Difference in 2-year Functional Outcomes Using Kinematic versus Mechanical Alignment in TKA: A Randomized Controlled Clinical Trial.
Neutral mechanical alignment (MA) in total knee arthroplasty (TKA) aims to position femoral and tibial components perpendicular to the mechanical axis of the limb. In contrast, kinematic alignment (KA) attempts to match implant position to the prearthritic anatomy of the individual patient with the aim of improving functional outcome. However, comparative data between the two techniques are lacking. In this randomized trial, we asked: (1) Are 2-year patient-reported outcome scores enhanced in patients with KA compared with an MA technique? (2) How does postoperative component alignment differ between the techniques? (3) Is the proportion of patients undergoing reoperation at 2 years different between the techniques? Ninety-nine primary TKAs in 95 patients were randomized to either MA (n = 50) or KA (n = 49) groups. A pilot study of 20 TKAs was performed before this trial using the same patient-specific guides positioning in kinematic alignment. In the KA group, patient-specific cutting blocks were manufactured using individual preoperative MRI data. In the MA group, computer navigation was used to ensure neutral mechanical alignment accuracy. Postoperative alignment was assessed with CT scan, and functional scores (including the Oxford Knee Score, WOMAC, and the Forgotten Joint Score) were assessed preoperatively and at 6 weeks, 6 months, and 1 and 2 years postoperatively. No patients were lost to followup. We set sample size at a minimum of 45 patients per treatment arm based on a 5-point improvement in the mean Oxford Knee Score (OKS; the previously reported minimum clinically significant difference for the OKS in TKA), a pooled SD of 8.3, 80% power, and a two-sided significance level of 5%. We observed no difference in 2-year change scores (postoperative minus preoperative score) in KA versus MA patients for the OKS (mean 21, SD 8 versus 20, SD 8, least square means 1.0, 95% confidence interval [CI], -1.4 to 3.4, p = 0.4), WOMAC score (mean 38, SD 18 versus 35, SD 8, least square means 3, 95% CI, -3.2 to 8.9, p = 0.3), or Forgotten Joint score (28 SD 37 versus 28, SD 28, least square means 0.8, 95% CI, -9.1-10.7, p = 0.8). Postoperative hip-knee-ankle axis was not different between groups (mean KA 0.4° varus SD 3.5 versus MA 0.7° varus SD 2.0), but in the KA group, the tibial component was a mean 1.9° more varus than the MA group (95% CI, 0.8°-3.0°, p = 0.003) and the femoral component in 1.6° more valgus (95% CI, -2.5° to -0.7°, p = 0.003). Complication rates were not different between groups. We found no difference in 2-year patient-reported outcome scores in TKAs implanted using the KA versus an MA technique. The theoretical advantages of improved pain and function that form the basis of the design rationale of KA were not observed in this study. Currently, it is unknown whether the alterations in component alignment seen with KA will compromise long-term survivorship of TKA. In this study, we were unable to demonstrate an advantage to KA in terms of pain or function that would justify this risk. Level I, therapeutic study.


Heritability of resistance to oxidative stress in early life.
Oxidative stress has recently been suggested to play an important role in life-history evolution, but little is known about natural variation and heritability of this physiological trait. Here, we explore phenotypic variation in resistance to oxidative stress of cross-fostered yellow-legged gull (Larus cachinnans) chicks. Resistance to oxidative stress was not related to plasma antioxidants at hatching, which are mostly derived from maternal investment into eggs. Common environmental effects on phenotypic variation in resistance to oxidative stress were not significant. Heritability was relatively low and nonsignificant in hatchlings, but interestingly, the chicks of age 8 days showed high and significant heritability (h(2) = 0.59). Our results suggest that resistance to oxidative stress is determined mainly by the genotype as chicks grow. Further work is required to explore the genetic role of oxidative stress in life-history evolution.


Improving accuracy of intraoperative diagnosis of endometriosis: Role of firefly in minimal access robotic surgery.
Endometriosis continues to be a major primary gynecologic etiology of chronic pelvic pain. The symptom profile, which includes cyclic pelvic pain, dysmenorrhea, and dyspareunia or dyschezia, is nonspecific and does not correlate with the extent or severity of disease. Trans-vaginal or trans-rectal ultrasound, as well as magnetic resonance imaging, can help visualize endometriomas and deeply infiltrating endometriosis. Additionally, there have been no serum marker tests available so far. However, even intraoperatively, the diagnosis may be missed, leading to under diagnosis and delayed or noninitiation of treatment. There are thought to be three distinct endometriotic lesions of the pelvis that are seen laparoscopically. The first is that which is visible on the pelvic peritoneal surface or the surface of the ovary, which is commonly termed peritoneal endometriosis. Second, endometriotic lesions that occur within the ovary and form cysts that are often lined with endometrioid mucosa are termed endometriomas. Lastly, rectovaginal endometriomas are endometriotic lesions that contain a mixture of adipose and fibrous tissue located between the rectum and vagina. All of these lesions can be singular or multiple and the pelvis may contain one or all three types of lesions. The shared histologic feature with all three lesions is the presence of endometrial epithelial cells or endometrial stroma. During a diagnostic procedure, the da-Vinci robot and its firefly mode allow for three dimensional visualization and seven degrees of instrument articulation for meticulous dissection of fibrotic areas of peritoneum that may contain deep infiltrating lesions of endometriosis. This case report describes a relatively new and innovative technique for effectively diagnosing and successfully treating endometriosis when other less invasive methods have failed.


Inhibitors of protein disulfide isomerase suppress apoptosis induced by misfolded proteins.
A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction and oxidation of disulfides. Through a small molecule screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated ER membranes and trigger apoptotic cell death via mitochondrial outer-membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon 1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI represents a new mechanism linking protein misfolding and apoptotic cell death.


Trends and correlates of the sex distribution among U.S. assisted reproductive technology births.
To assess national trends in the sex distribution of live-born infants in the assisted reproductive technology (ART) and general population and to identify factors correlated with offspring sex. Retrospective cohort study. Fertility treatment centers. All live-born infants included in the National Vital Statistics System and resulting from ART cycles reported to the National ART Surveillance System during 2006-14. Not applicable. Trends in the proportion of male infants in the general population and proportion of males from fresh ART cycles among all ART live-born infants and singletons after single ET. There were 214,274 live-born infants resulting from fresh ART cycles; 53.5% (5,492/10,266) of infants resulting from PGD/PGS cycles were male, as compared with 50.6% (103,228/204,008) in the non-PGD/PGS group. Among non-PGD/PGS cycles, blastocyst transfer was positively associated with male infants (adjusted risk ratio [aRR] = 1.03; 95% confidence interval [CI], 1.02-1.04). Intracytoplasmic sperm injection was negatively associated with male infants (aRR = 0.94; 95% CI, 0.93-0.95) and for singletons after single ET (aRR = 0.93; 95% CI, 0.90-0.95), as was transfer of two embryos (aRR 0.98; 95% CI, 0.97-0.99) or three or more embryos (aRR = 0.98; 95% CI, 0.96-0.99) among all live births from cycles without PGD/PGS use. The proportion of male live-born infants among ART population did not change during 2006-14, ranging from 50.5% to 51.2%. Factors such as blastocyst transfer, intracytoplasmic sperm injection use, embryo stage, and number of embryos transferred may be associated with infant sex; further investigation is needed to understand possible underlying causes.


Development of calpain-specific inactivators by screening of positional scanning epoxide libraries.
Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.


Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma.
Although the prognostic value of B7-H1 and B7-H4 expression by tumor cells in clear cell renal cell carcinoma (ccRCC) has been established, the role of B7-H3 is unknown. As such, we evaluated the association of B7-H3 expression with clinicopathologic outcomes in patients treated for ccRCC. Nephrectomy specimens from 743 consecutive patients treated for ccRCC at our institution from 1990 to 1999 were evaluated for B7-H3 expression by immunohistochemical staining. Associations of B7-H3 expression with clinical and pathologic features were evaluated using chi2 and Fisher's exact tests. Associations of B7-H3 expression with death from RCC were evaluated using Cox proportional hazards regression models. B7-H3 expression by tumor cells or tumor vasculature was noted in 17% and 95% of specimens, respectively. The presence of either tumor cell or diffuse tumor vasculature expression of B7-H3 was present in 46% of specimens and was associated with multiple adverse clinical and pathologic features. After multivariable adjustment, the presence of either tumor cell or diffuse tumor vasculature B7-H3 expression was significantly associated with an increased risk of death from RCC (risk ratio, 1.38; 95% confidence interval, 1.03-1.84; P = 0.029). Both tumor cell and tumor vasculature B7-H3 expression convey important information to predict ccRCC outcomes. Collectively, our past and present studies pertaining to B7-H ligand expression indicate that ccRCC may use redundant mechanisms to compromise host antitumoral immunity. Future studies will focus on the effect of combined B7-H ligand expression in RCC.


Altered Oscillatory Dynamics of CA1 Parvalbumin Basket Cells during Theta-Gamma Rhythmopathies of Temporal Lobe Epilepsy.
Recent reports in human demonstrate a role of theta-gamma coupling in memory for spatial episodes and a lack of coupling in people experiencing temporal lobe epilepsy, but the mechanisms are unknown. Using multisite silicon probe recordings of epileptic rats engaged in episodic-like object recognition tasks, we sought to evaluate the role of theta-gamma coupling in the absence of epileptiform activities. Our data reveal a specific association between theta-gamma (30-60 Hz) coupling at the proximal stratum radiatum of CA1 and spatial memory deficits. We targeted the microcircuit mechanisms with a novel approach to identify putative interneuronal types in tetrode recordings (parvalbumin basket cells in particular) and validated classification criteria in the epileptic context with neurochemical identification of intracellularly recorded cells. In epileptic rats, putative parvalbumin basket cells fired poorly modulated at the falling theta phase, consistent with weaker inputs from Schaffer collaterals and attenuated gamma oscillations, as evaluated by theta-phase decomposition of current-source density signals. We propose that theta-gamma interneuronal rhythmopathies of the temporal lobe are intimately related to episodic memory dysfunction in this condition.


Safety of Immediate Posterior Trunk Soft-Tissue Reconstruction in Older Adults.
Older patients, especially those older than 65 years, are accounting for an ever-increasing share of hospital costs, especially surgical procedures. Studies regarding the safety of surgery in these patients have been widespread in the past decade. Despite these efforts, there is a paucity of information regarding the safety of reconstruction following spinal surgery in older patients. We conducted a retrospective cohort study of patients undergoing soft-tissue reconstruction of the posterior trunk after oncologic spine surgery. Demographic, medical, and surgical variables were measured. The primary outcome variable was the development of a medical or surgical complication postoperatively. Other secondary outcomes included development of a medical complication alone, specific surgical complications (seroma, hematoma, infection, wound dehiscence, and (cerebrospinal fluid leak), unplanned reoperation, and 60-day operative mortality. A priori power analysis suggested 205 cases would be needed. The study included 286 cases in 256 patients. The cohorts were similar in terms of demographic, surgical, and medical variables, though the older cohort had higher mean American Society of Anesthesiologists score (2.09 versus 1.65; P < 0.0001). We found no correlation between increasing age and increased rates of medical or surgical complications (35.9% for older patients versus 44.7% for younger patients; P = 0.31). However, mean American Society of Anesthesiologists score did correlate with complications (1.88 for patients with complications versus 1.69 for patients without; P = 0.04). We demonstrated no increased risk for complications among older patients. Complex soft-tissue reconstruction of the posterior trunk is safe in elderly patients undergoing oncologic spinal surgery.


In situ imaging reveals the biomass of giant protists in the global ocean.
Planktonic organisms play crucial roles in oceanic food webs and global biogeochemical cycles. Most of our knowledge about the ecological impact of large zooplankton stems from research on abundant and robust crustaceans, and in particular copepods. A number of the other organisms that comprise planktonic communities are fragile, and therefore hard to sample and quantify, meaning that their abundances and effects on oceanic ecosystems are poorly understood. Here, using data from a worldwide in situ imaging survey of plankton larger than 600 μm, we show that a substantial part of the biomass of this size fraction consists of giant protists belonging to the Rhizaria, a super-group of mostly fragile unicellular marine organisms that includes the taxa Phaeodaria and Radiolaria (for example, orders Collodaria and Acantharia). Globally, we estimate that rhizarians in the top 200 m of world oceans represent a standing stock of 0.089 Pg carbon, equivalent to 5.2% of the total oceanic biota carbon reservoir. In the vast oligotrophic intertropical open oceans, rhizarian biomass is estimated to be equivalent to that of all other mesozooplankton (plankton in the size range 0.2-20 mm). The photosymbiotic association of many rhizarians with microalgae may be an important factor in explaining their distribution. The previously overlooked importance of these giant protists across the widest ecosystem on the planet changes our understanding of marine planktonic ecosystems.


Flashing light emitting diodes (LEDs) induce proteins, polyunsaturated fatty acids and pigments in three microalgae.
As the periodic emission of light pulses by light emitting diodes (LEDs) is known to stimulate growth or induce high value biocompounds in microalgae, this flashing light regime was tested on growth and biochemical composition of the microalgae Nannochloropsis gaditana, Koliella antarctica and Tetraselmis chui. At low flashing light frequencies (e.g., 5 and 50 Hz, Duty cycle = 0.05), a strain-dependent growth inhibition and an accumulation of protein, polyunsaturated fatty acids, chlorophyll or carotenoids (lutein, β-carotene, violaxanthin and neoxanthin) was observed. In addition, a 4-day application of low-frequency flashing light to concentrated cultures increased productivities of eicosapentaenoic acid (EPA) and specific carotenoids up to three-fold compared to continuous or high frequency flashing light (500 Hz, Duty cycle = 0.05). Therefore, applying low-frequency flashing light as finishing step in industrial production can increase protein, polyunsaturated fatty acids or pigment contents in biomass, leading to high-value algal products.


Peripheral Blood Mononuclear Cells and Platelets Mitochondrial Dysfunction, Oxidative Stress, and Circulating mtDNA in Cardiovascular Diseases.
Cardiovascular diseases (CVDs) are devastating disorders and the leading cause of mortality worldwide. The pathophysiology of cardiovascular diseases is complex and multifactorial and, in the past years, mitochondrial dysfunction and excessive production of reactive oxygen species (ROS) have gained growing attention. Indeed, CVDs can be considered as a systemic alteration, and understanding the eventual implication of circulating blood cells peripheral blood mononuclear cells (PBMCs) and or platelets, and particularly their mitochondrial function, ROS production, and mitochondrial DNA (mtDNA) releases in patients with cardiac impairments, appears worthwhile. Interestingly, reports consistently demonstrate a reduced mitochondrial respiratory chain oxidative capacity related to the degree of CVD severity and to an increased ROS production by PBMCs. Further, circulating mtDNA level was generally modified in such patients. These data are critical steps in term of cardiac disease comprehension and further studies are warranted to challenge the possible adjunct of PBMCs' and platelets' mitochondrial dysfunction, oxidative stress, and circulating mtDNA as biomarkers of CVD diagnosis and prognosis. This new approach might also allow further interesting therapeutic developments.


RAS testing in metastatic colorectal cancer: advances in Europe.
Personalized medicine shows promise for maximizing efficacy and minimizing toxicity of anti-cancer treatment. KRAS exon 2 mutations are predictive of resistance to epidermal growth factor receptor-directed monoclonal antibodies in patients with metastatic colorectal cancer. Recent studies have shown that broader RAS testing (KRAS and NRAS) is needed to select patients for treatment. While Sanger sequencing is still used, approaches based on various methodologies are available. Few CE-approved kits, however, detect the full spectrum of RAS mutations. More recently, "next-generation" sequencing has been developed for research use, including parallel semiconductor sequencing and reversible termination. These techniques have high technical sensitivities for detecting mutations, although the ideal threshold is currently unknown. Finally, liquid biopsy has the potential to become an additional tool to assess tumor-derived DNA. For accurate and timely RAS testing, appropriate sampling and prompt delivery of material is critical. Processes to ensure efficient turnaround from sample request to RAS evaluation must be implemented so that patients receive the most appropriate treatment. Given the variety of methodologies, external quality assurance programs are important to ensure a high standard of RAS testing. Here, we review technical and practical aspects of RAS testing for pathologists working with metastatic colorectal cancer tumor samples. The extension of markers from KRAS to RAS testing is the new paradigm for biomarker testing in colorectal cancer.


Outcome measures in ANCA-associated vasculitis.
The outcome in patients with antineutrophil cytoplasm antibody (ANCA)-associated vasculitis has considerably improved with the use of potent immunosuppression. In the most severe cases, mortality remains an important risk despite aggressive intervention. However, for most patients who survive, quality of survival is affected by morbidity due to low-grade disease activity, episodes of relapse, damage from the effects of disease and its treatment, development of associated comorbidity, and the social and psychological problems of chronic disease. A rational approach to management of patients with ANCA-associated vasculitis requires careful measurement of these different facets of disease, so that treatment is appropriate. This article reviews the development of assessment tools used to quantify disease in vasculitis, which have been extensively used in clinical trials and are also appropriate for use in individual patient care.


DGGE-based detection method for Quahog Parasite Unknown (QPX).
Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.


Variability in the use of pulse oximeters with children in Kenyan hospitals: A mixed-methods analysis.
Pulse oximetry, a relatively inexpensive technology, has the potential to improve health outcomes by reducing incorrect diagnoses and supporting appropriate treatment decisions. There is evidence that in low- and middle-income countries, even when available, widespread uptake of pulse oximeters has not occurred, and little research has examined why. We sought to determine when and with which children pulse oximeters are used in Kenyan hospitals, how pulse oximeter use impacts treatment provision, and the barriers to pulse oximeter use. We analyzed admissions data recorded through Kenya's Clinical Information Network (CIN) between September 2013 and February 2016. We carried out multiple imputation and generated multivariable regression models in R. We also conducted interviews with 30 healthcare workers and staff from 14 Kenyan hospitals to examine pulse oximetry adoption. We adapted the Integrative Model of Behavioural Prediction to link the results from the multivariable regression analyses to the qualitative findings. We included 27,906 child admissions from 7 hospitals in the quantitative analyses. The median age of the children was 1 year, and 55% were male. Three-quarters had a fever, over half had a cough; other symptoms/signs were difficulty breathing (34%), difficulty feeding (34%), and indrawing (32%). The most common diagnoses were pneumonia, diarrhea, and malaria: 45%, 35%, and 28% of children, respectively, had these diagnoses. Half of the children obtained a pulse oximeter reading, and of these, 10% had an oxygen saturation level below 90%. Children were more likely to receive a pulse oximeter reading if they were not alert (odds ratio [OR]: 1.30, 95% confidence interval (CI): 1.09, 1.55, p = 0.003), had chest indrawing (OR: 1.28, 95% CI: 1.17, 1.40, p < 0.001), or a very high respiratory rate (OR: 1.27, 95% CI: 1.13, 1.43, p < 0.001), as were children admitted to certain hospitals, at later time periods, and when a Paediatric Admission Record (PAR) was used (OR PAR used compared with PAR not present: 2.41, 95% CI: 1.98, 2.94, p < 0.001). Children were more likely to be prescribed oxygen if a pulse oximeter reading was obtained (OR: 1.42, 95% CI:1.25, 1.62, p < 0.001) and if this reading was below 90% (OR: 3.29, 95% CI: 2.82, 3.84, p < 0.001). The interviews indicated that the main barriers to pulse oximeter use are inadequate supply, broken pulse oximeters, and insufficient training on how, when, and why to use pulse oximeters and interpret their results. According to the interviews, variation in pulse oximeter use between hospitals is because of differences in pulse oximeter availability and the leadership of senior doctors in advocating for pulse oximeter use, whereas variation within hospitals over time is due to repair delays. Pulse oximeter use increased over time, likely because of the CIN's feedback to hospitals. When pulse oximeters are used, they are sometimes used incorrectly and some healthcare workers lack confidence in readings that contradict clinical signs. The main limitations of the study are that children with high levels of missing data were not excluded, interview participants might not have been representative, and the interviews did not enable a detailed exploration of differences between counties or across senior management groups. There remain major challenges to implementing pulse oximetry-a cheap, decades old technology-into routine care in Kenya. Implementation requires efficient and transparent procurement and repair systems to ensure adequate availability. Periodic training, structured clinical records that include prompts, the promotion of pulse oximetry by senior doctors, and monitoring and feedback might also support pulse oximeter use. Our findings can inform strategies to support the use of pulse oximeters to guide prompt and effective treatment, in line with the Sustainable Development Goals. Without effective implementation, the potential benefits of pulse oximeters and possible hospital cost-savings by targeting oxygen therapy might not be realized.


Wound healing in the intestinal wall. A comparison between experimental ileal and colonic anastomoses.
The healing of ileal and colonic anastomoses is compared in rabbits. The intestinal segment that contains the anastomosis shows a temporary loss of strength, which is reflected in a decreased bursting pressure. This loss of strength is accompanied by a massive loss of collagen, measured as hydroxyproline, both in ileum and in colon. In ileum, hydroxyproline concentrations, expressed on a dry weight basis, are lowered by 30 per cent, one day after operation. Thereafter, they rise again, after seven days reaching a level that is 40 per cent enhanced as compared with unwounded tissue. Maximal decrease in colon, measured two days after operation, is 40 per cent. After seven days hydroxyproline levels are back at preoperative values. In colon, significant loss of hydroxyproline is also apparent in the intestinal segment proximal to the anastomosis. This phenomenon does not occur in ileum. These results clearly demonstrate that after ileal anastomosis a loss of collagen occurs similar to that in colonic anastomoses. The fact that the loss of collagen is less extensive and more rapidly restored may be important in explaining the lesser incidence of leakage encountered after surgery of the small intestine.


Continuity of primary care: to whom does it matter and when?
Inconsistent findings on the value of continuity of care can stem from variability in its importance to different subsets of patients. We therefore examined the association among patient and visit characteristics and extent to which the patient valued continuity of care (PVC). We hypothesized that continuity would be more important to patients who are older, sicker, and female, who have established a relationship with their physician, and whose visit addresses more complex problems. A study of 4,454 consecutive outpatient visits to 138 community-based family physicians used a 3-item measure (alpha = 0.67) of PVC. The patient's report of (1) the adequacy of primary care for the visit and (2) satisfaction with the physician on that visit was assessed with multiple measures. Analyses examined the associations among PVC and patient-reported satisfaction with the physician and adequacy of the visit. Extremes of age, female sex, less education, Medicare and Medicaid insurance, number of chronic conditions and medications, number of visits to the practice, and worse self-reported health status were associated with higher value placed on continuity (P < .001 for all except sex, where P = .015). Patients who value continuity and did not see a regular physician rated adequacy of the visit lower (for 7 attributes of the visit) than those seeing their own physician. Satisfaction with the physician for the visit was greatest among patients who value continuity and saw their regular physician. Continuity of physician care is associated with more positive assessments of the visit and appears to be particularly important for more vulnerable patients. Health care systems and primary care practices should devote additional effort to maintaining a continuity relationship with these vulnerable patients.


ON THE PERMANENT LIFE OF TISSUES OUTSIDE OF THE ORGANISM.
In two series of experiments made at the end of 1911 and at the beginning of 1912, new techniques were developed with the view of investigating the problem of prolonging indefinitely the life of tissues isolated from the organism. These techniques are far from perfect and will doubtless be modified in the future. But they have already permitted the establishment of new facts. Fragments of connective tissue have been kept in vitro in a condition of active life for more than two months. As a few cultures are now eighty-five days old and are growing very actively, it is probable that, if no accident occurs, the life of these cultures will continue for a long time. In some cases the rate of growth of the tissues increased in direct ratio to the age of the culture. Fragments of heart pulsated rhythmically at the beginning of the third month of their life in vitro. These facts show that experiments made with these or with more perfect techniques and followed over long periods of time may lead to the solution of the problem of permanent life of tissues in vitro, and give important information on the characters acquired by tissues liberated from the control of the organism from which they were derived.


Non-fullerene electron acceptors for use in organic solar cells.
The active layer in a solution processed organic photovoltaic device comprises a light absorbing electron donor semiconductor, typically a polymer, and an electron accepting fullerene acceptor. Although there has been huge effort targeted to optimize the absorbing, energetic, and transport properties of the donor material, fullerenes remain as the exclusive electron acceptor in all high performance devices. Very recently, some new non-fullerene acceptors have been demonstrated to outperform fullerenes in comparative devices. This Account describes this progress, discussing molecular design considerations and the structure-property relationships that are emerging. The motivation to replace fullerene acceptors stems from their synthetic inflexibility, leading to constraints in manipulating frontier energy levels, as well as poor absorption in the solar spectrum range, and an inherent tendency to undergo postfabrication crystallization, resulting in device instability. New acceptors have to address these limitations, providing tunable absorption with high extinction coefficients, thus contributing to device photocurrent. The ability to vary and optimize the lowest unoccupied molecular orbital (LUMO) energy level for a specific donor polymer is also an important requirement, ensuring minimal energy loss on electron transfer and as high an internal voltage as possible. Initially perylene diimide acceptors were evaluated as promising acceptor materials. These electron deficient aromatic molecules can exhibit good electron transport, facilitated by close packed herringbone crystal motifs, and their energy levels can be synthetically tuned. The principal drawback of this class of materials, their tendency to crystallize on too large a length scale for an optimal heterojunction nanostructure, has been shown to be overcome through introduction of conformation twisting through steric effects. This has been primarily achieved by coupling two units together, forming dimers with a large intramolecular twist, which suppresses both nucleation and crystal growth. The generic design concept of rotationally symmetrical aromatic small molecules with extended π orbital delocalization, including polyaromatic hydrocarbons, phthalocyanines, etc., has also provided some excellent small molecule acceptors. In most cases, additional electron withdrawing functionality, such as imide or ester groups, can be incorporated to stabilize the LUMO and improve properties. New calamitic acceptors have been developed, where molecular orbital hybridization of electron rich and poor segments can be judiciously employed to precisely control energy levels. Conformation and intermolecular associations can be controlled by peripheral functionalization leading to optimization of crystallization length scales. In particular, the use of rhodanine end groups, coupled electronically through short bridged aromatic chains, has been a successful strategy, with promising device efficiencies attributed to high lying LUMO energy levels and subsequently large open circuit voltages.


The WNK-SPAK/OSR1 pathway: master regulator of cation-chloride cotransporters.
The WNK-SPAK/OSR1 kinase complex is composed of the kinases WNK (with no lysine) and SPAK (SPS1-related proline/alanine-rich kinase) or the SPAK homolog OSR1 (oxidative stress-responsive kinase 1). The WNK family senses changes in intracellular Cl(-) concentration, extracellular osmolarity, and cell volume and transduces this information to sodium (Na(+)), potassium (K(+)), and chloride (Cl(-)) cotransporters [collectively referred to as CCCs (cation-chloride cotransporters)] and ion channels to maintain cellular and organismal homeostasis and affect cellular morphology and behavior. Several genes encoding proteins in this pathway are mutated in human disease, and the cotransporters are targets of commonly used drugs. WNKs stimulate the kinases SPAK and OSR1, which directly phosphorylate and stimulate Cl(-)-importing, Na(+)-driven CCCs or inhibit the Cl(-)-extruding, K(+)-driven CCCs. These coordinated and reciprocal actions on the CCCs are triggered by an interaction between RFXV/I motifs within the WNKs and CCCs and a conserved carboxyl-terminal docking domain in SPAK and OSR1. This interaction site represents a potentially druggable node that could be more effective than targeting the cotransporters directly. In the kidney, WNK-SPAK/OSR1 inhibition decreases epithelial NaCl reabsorption and K(+) secretion to lower blood pressure while maintaining serum K(+). In neurons, WNK-SPAK/OSR1 inhibition could facilitate Cl(-) extrusion and promote γ-aminobutyric acidergic (GABAergic) inhibition. Such drugs could have efficacy as K(+)-sparing blood pressure-lowering agents in essential hypertension, nonaddictive analgesics in neuropathic pain, and promoters of GABAergic inhibition in diseases associated with neuronal hyperactivity, such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism.


Diabetes in urban African-Americans. XIX. Prediction of the need for pharmacological therapy.
To develop a prediction rule that will identify patients who will require pharmacological therapy within 6 months of first presentation to a diabetes clinic. Among the patients who came to the Grady Diabetes Clinic between 1991 and 1997, we randomized 557 frequent attenders to a development group and 520 frequent attenders to a validation group. Using multiple logistical regression, we derived a prediction rule in the development group to project whether patients would require pharmacological intervention to achieve HbA1c levels <7% after 6 months. The utility of the prediction rule was then confirmed in the validation group and tested prospectively on an additional group of 93 patients who presented from 1997 to 1998. Performance of the prediction rule was assessed using receiver operating characteristic (ROC) curves. The rule (-4.469 + 1.932 x sulfonylurea Rx + 1.334 x insulin Rx + 0.196 x duration + 0.468 x fasting glucose, where "Rx" indicates a prescription) predicted the need for pharmacological intervention in the development group (P < 0.0001). Use of insulin or sulfonylurea therapy at presentation, duration of diabetes, and fasting glucose levels were significant predictors of the future need for pharmacological management. The prediction rule also performed well in the validation group (positive predictive value 90%, correlation between predicted and observed need for medical management 0.99). ROC curves confirmed the value of the prediction rule (area under the curves was 0.91 for the development group, 0.85 for the validation group, and 0.81 for the prospective group). Early identification of individuals who will require pharmacological intervention to achieve national standards for glycemic control can be achieved with high probability, thus allowing for more efficient management of diabetes.


Clinical efficacy of the microfracture technique for articular cartilage repair in the knee: an evidence-based systematic analysis.
Despite the popularity of microfracture as a first-line treatment for articular cartilage defects in the knee, systematic information on its clinical efficacy for articular cartilage repair and long-term improvement of knee function is not available. Systematic analysis of the existing clinical literature of microfracture in the knee can improve the understanding of the advantages and limitations of this cartilage repair technique and can help to optimize its indications and clinical outcomes. Systematic review. A comprehensive literature search was performed using established search engines (MEDLINE, EMBASE, CINAHL, Cochrane Central Register of Controlled Trials) to identify original human studies of articular cartilage repair with microfracture. Modified Coleman Methodology Scores were used to analyze the quality of the existing studies. Clinical efficacy of articular cartilage repair was evaluated by systematic analysis of short- and long-term functional outcome scores, macroscopic and microscopic repair cartilage quality, and findings of postoperative magnetic resonance imaging. Twenty-eight studies describing 3122 patients were included in the review. The average follow-up was 41 months, with only 5 studies reporting follow-up of 5 years or more. Six studies were randomized controlled trials and the mean Coleman Methodology Score was 58 (range, 22-97). Microfracture effectively improved knee function in all studies during the first 24 months after microfracture, but the reports on durability of the initial functional improvement were conflicting. Several factors were identified that affected clinical outcome. Defect fill on magnetic resonance imaging was highly variable and correlated with functional outcome. Macroscopic repair cartilage quality positively affected long-term failure rate, while the influence of histologic repair tissue quality remained inconclusive. This systematic analysis shows that microfracture provides effective short-term functional improvement of knee function but insufficient data are available on its long-term results. Shortcomings of the technique include limited hyaline repair tissue, variable repair cartilage volume, and possible functional deterioration. The quality of the currently available data on micro-fracture is still limited by the variability of results and study designs. Further well-designed studies are needed to determine the long-term efficacy of microfracture and to define its specific clinical indications compared to other cartilage repair techniques.


Folate derived from cecal bacterial fermentation does not increase liver folate stores in 28-d folate-depleted male Sprague-Dawley rats.
This study assessed the ability of rats to absorb and store the folate synthesized by cecal bacteria. Male weanling Sprague-Dawley rats were folate depleted by feeding a low folacin AIN93G formulated basal diet for 28 d; they were then fed repletion diets containing folate (0.25-1.0 mg/kg diet), dietary fiber (DF; wheat bran, oat bran, ground corn, wheat germ) or undigested and fermented dietary material (UFDM; polydextrose, inulin) in the presence and absence of an antibiotic (succinylsulfathiazole). Fermentation was stimulated by DF and UFDM and reduced by the antibiotic. In the absence of succinylsulfathiazole, the increase in liver folate (during the repletion phase) was proportional only to the folate content of the diet and did not vary with added DF or UFDM. Adding succinylsulfathiazole lowered total folate excretion from 13.8 +/- 8.2 to 4.8 +/- 2.9 nmol/d (pooled diets, P < 0.00001) in agreement with its role in inhibiting bacterial folate synthesis. In addition, succinylsulfathiazole lowered liver folate in rats fed control and test diets approximately equally with a mean decrease from 11.6 +/- 2.5 to 7.5 +/- 2.5 nmol/g wet liver (pooled diets, P < 0.00001), suggesting that the antibiotic also affected rat folate absorption and/or metabolism. Increased bacterial fermentation and excretion as well as increased bacterial folate production in the presence of added DF and UFDM were demonstrated by increased volatile fatty acid content in cecal and fecal samples (P < 0.000001) and increased diaminopimelic acid, muramic acid and folate in feces (P < 0.00001). The magnitude of these changes depended on the type of DF and UFDM. These results show that bacterially synthesized folate is not substantially absorbed and stored in the liver of Sprague-Dawley male rats.


Network Firewall Dynamics and the Subsaturation Stabilization of HIV.
In 2001, Friedman et al. conjectured the existence of a "firewall effect" in which individuals who are infected with HIV, but remain in a state of low infectiousness, serve to prevent the virus from spreading. To evaluate this historical conjecture, we develop a new graph-theoretic measure that quantifies the extent to which Friedman's firewall hypothesis(FH)holds in a risk network. We compute this new measure across simulated trajectories of a stochastic discrete dynamical system that models a social network of 25,000 individuals engaging in risk acts over a period of 15 years. The model's parameters are based on analyses of data collected in prior studies of the real-world risk networks of people who inject drugs (PWID) in New York City. Analysis of system trajectories reveals the structural mechanisms by which individuals with mature HIV infections tend to partition the network into homogeneous clusters (with respect to infection status) and how uninfected clusters remain relatively stable (with respect to infection status) over long stretches of time. We confirm the spontaneous emergence of network firewalls in the system and reveal their structural role in the nonspreading of HIV.


Risk factors associated with acute exacerbation of chronic obstructive pulmonary disease in HIV-infected and uninfected patients.
To determine the association between HIV infection and other risk factors for acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Longitudinal, national Veterans Aging Cohort Study including 43 618 HIV-infected and 86 492 uninfected veterans. AECOPD was defined as an inpatient or outpatient COPD ICD-9 diagnosis accompanied by steroid and/or antibiotic prescription within 5 days. We calculated incidence rate ratios (IRR) and 95% confidence intervals (CI) for first AECOPD over 2 years and used Poisson regression models to adjust for risk factors. Over 234 099 person-years of follow-up, 1428 HIV-infected and 2104 uninfected patients had at least one AECOPD. HIV-infected patients had an increased rate of AECOPD compared with uninfected (18.8 vs. 13.3 per 1000 person-years, P < 0.001). In adjusted models, AECOPD risk was greater in HIV-infected individuals overall (IRR 1.54; 95% CI 1.44-1.65), particularly in those with more severe immune suppression when stratified by CD4 cell count (cells/μl) compared with uninfected (HIV-infected CD4 < 200: IRR 2.30, 95% CI 2.10-2.53, HIV-infected CD4 ≥ 200-349: IRR 1.32, 95% CI 1.15-1.51, HIV-infected CD4 ≥ 350: IRR 0.99, 95% CI 0.88-1.10). HIV infection also modified the association between current smoking and alcohol-related diagnoses with risk for AECOPD such that interaction terms for HIV and current smoking or HIV and alcohol-related diagnoses were each significantly associated with AECOPD. HIV infection, especially with lower CD4 cell count, is an independent risk factor for AECOPD. Enhanced susceptibility to harm from current smoking or unhealthy alcohol use in HIV-infected patients may also contribute to the greater rate of AECOPD.


Long Noncoding RNA POU3F3 and α-Synuclein in Plasma L1CAM Exosomes Combined with β-Glucocerebrosidase Activity: Potential Predictors of Parkinson's Disease.
Long noncoding RNAs (lncRNAs) are implicated in the autophagic-lysosomal pathway (ALP) and are closely linked to Parkinson's disease (PD) pathology. β-Glucocerebrosidase (GCase) has also been reported to be correlated with α-synuclein (α-syn) proteostasis. However, lncRNAs and α-syn in neural-derived L1CAM exosomes and GCase activity in the plasma of PD patients have not been studied. This study used an ultrasensitive methodology, fluorescence nanoparticle tracking analysis (NTA), to measure plasma L1CAM exosomes and Quanterix Simoa to measure α-syn concentrations in L1CAM exosomes. Eighty-five healthy controls and 93 PD patients were enrolled, and several scales were used to rate the severity of PD. Receiver operating characteristic (ROC) curves were applied to map the diagnostic accuracy of categorizing PD patients and healthy subjects. We found increased Linc-POU3F3 and α-syn concentrations in L1CAM exosomes and decreased GCase activity in PD patients compared with controls. The three biomarkers displayed obvious differences among PD patients based on gender, H-Y stage, and UPDRS-III distribution. Interestingly, Linc-POU3F3 was significantly positively correlated with α-syn in L1CAM exosomes and inversely correlated with GCase activity in PD patients. Significant correlations were observed among L1CAM exosomal Linc-POU3F3 levels, GCase activity, and PD severity, including motor/cognitive dysfunction. Additionally, the combination of Linc-POU3F3 and α-syn in L1CAM exosomes and GCase activity could discriminate PD patients from controls. These results suggest that L1CAM exosomal Linc-POU3F3, L1CAM exosomal α-syn, and GCase activity may shed light on the mechanism underlying the autophagic-lysosomal system in the pathogenesis of PD and could be used to assess the severity of PD.


Ethnic inequalities and pathways to care in psychosis in England: a systematic review and meta-analysis.
As part of a national programme to tackle ethnic inequalities, we conducted a systematic review and meta-analysis of research on ethnic inequalities in pathways to care for adults with psychosis living in England and/or Wales. Nine databases were searched from inception to 03.07.17 for previous systematic reviews, including forward and backward citation tracking and a PROSPERO search to identify ongoing reviews. We then carried forward relevant primary studies from included reviews (with the latest meta-analyses reporting on research up to 2012), supplemented by a search on 18.10.17 in MEDLINE, Embase, PsycINFO and CINAHL for primary studies between 2012 and 2017 that had not been covered by previous meta-analyses. Forty studies, all conducted in England, were included for our updated meta-analyses on pathways to care. Relative to the White reference group, elevated rates of civil detentions were found for Black Caribbean (OR = 3.43, 95% CI = 2.68 to 4.40, n = 18), Black African (OR = 3.11, 95% CI = 2.40 to 4.02, n = 6), and South Asian patients (OR = 1.50, 95% CI 1.07 to 2.12, n = 10). Analyses of each Mental Health Act section revealed significantly higher rates for Black people under (civil) Section 2 (OR = 1.53, 95% CI = 1.11 to 2.11, n = 3). Rates in repeat admissions were significantly higher than in first admission for South Asian patients (between-group difference p < 0.01). Some ethnic groups had more police contact (Black African OR = 3.60, 95% CI = 2.15 to 6.05, n = 2; Black Caribbean OR = 2.64, 95% CI = 1.88 to 3.72, n = 8) and criminal justice system involvement (Black Caribbean OR = 2.76, 95% CI = 2.02 to 3.78, n = 5; Black African OR = 1.92, 95% CI = 1.32 to 2.78, n = 3). The White Other patients also showed greater police and criminal justice system involvement than White British patients (OR = 1.49, 95% CI = 1.03 to 2.15, n = 4). General practitioner involvement was less likely for Black than the White reference group. No significant variations over time were found across all the main outcomes. Our updated meta-analyses reveal persisting but not significantly worsening patterns of ethnic inequalities in pathways to psychiatric care, particularly affecting Black groups. This provides a comprehensive evidence base from which to inform policy and practice amidst a prospective Mental Health Act reform. CRD42017071663.


Photoacoustic Neuroimaging - Perspectives on a Maturing Imaging Technique and its Applications in Neuroscience.
A prominent goal of neuroscience is to improve our understanding of how brain structure and activity interact to produce perception, emotion, behavior, and cognition. The brain's network activity is inherently organized in distinct spatiotemporal patterns that span scales from nanometer-sized synapses to meter-long nerve fibers and millisecond intervals between electrical signals to decades of memory storage. There is currently no single imaging method that alone can provide all the relevant information, but intelligent combinations of complementary techniques can be effective. Here, we thus present the latest advances in biomedical and biological engineering on photoacoustic neuroimaging in the context of complementary imaging techniques. A particular focus is placed on recent advances in whole-brain photoacoustic imaging in rodent models and its influential role in bridging the gap between fluorescence microscopy and more non-invasive techniques such as magnetic resonance imaging (MRI). We consider current strategies to address persistent challenges, particularly in developing molecular contrast agents, and conclude with an overview of potential future directions for photoacoustic neuroimaging to provide deeper insights into healthy and pathological brain processes.


Are we close to defining a metabolomic signature of human obesity? A systematic review of metabolomics studies.
Obesity is a disorder characterized by a disproportionate increase in body weight in relation to height, mainly due to the accumulation of fat, and is considered a pandemic of the present century by many international health institutions. It is associated with several non-communicable chronic diseases, namely, metabolic syndrome, type 2 diabetes mellitus (T2DM), cardiovascular diseases (CVD), and cancer. Metabolomics is a useful tool to evaluate changes in metabolites due to being overweight and obesity at the body fluid and cellular levels and to ascertain metabolic changes in metabolically unhealthy overweight and obese individuals (MUHO) compared to metabolically healthy individuals (MHO). We aimed to conduct a systematic review (SR) of human studies focused on identifying metabolomic signatures in obese individuals and obesity-related metabolic alterations, such as inflammation or oxidative stress. We reviewed the literature to identify studies investigating the metabolomics profile of human obesity and that were published up to May 7th, 2019 in SCOPUS and PubMed through an SR. The quality of reporting was evaluated using an adapted of QUADOMICS. Thirty-three articles were included and classified according to four types of approaches. (i) studying the metabolic signature of obesity, (ii) studying the differential responses of obese and non-obese subjects to dietary challenges (iii) studies that used metabolomics to predict weight loss and aimed to assess the effects of weight loss interventions on the metabolomics profiles of overweight or obese human subjects (iv) articles that studied the effects of specific dietary patterns or dietary compounds on obesity-related metabolic alterations in humans. The present SR provides state-of-the-art information about the use of metabolomics as an approach to understanding the dynamics of metabolic processes involved in human obesity and emphasizes metabolic signatures related to obesity phenotypes.


Genetic and physical maps of the Alcaligenes eutrophus CH34 megaplasmid pMOL28 and its derivative pMOL50 obtained after temperature-induced mutagenesis and mortality.
We describe the construction of restriction and genetic maps of plasmid pMOL28, which has a size of approximately 180 kb. To do so, partial BamHI-digested DNA of pMOL28 was cloned into cosmid pLAFR3, which can package up to 20-30 kb inserted DNA. Subsequently, a cosmid walking strategy, combined with BamHI or EcoRI restriction analysis and hybridization, was used to construct the restriction maps for both enzymes. On these maps, 35 BamHI fragments and 29 EcoRI fragments were placed, accounting for a total size of approximately 180 kb. We also analyzed several rearranged derivatives of pMOL28 that were obtained after a process of temperature-induced mortality and mutagenesis (TIMM), which is characteristic for Alcaligenes eutrophus CH34 and related strains. The restriction and genetic maps of pMOL50 (222 kb), an enlarged derivative of pMOL28 obtained after TIMM, were constructed. By comparing the pMOL28 and pMOL50 maps, at least two transposable elements were identified which participated in the formation of pMOL50 from pMOL28 during TIMM. These transposable elements were IS1086, which was recently sequenced, and a new element named IS1089, which is located on the 44-kb inserted DNA fragment in pMOL50. Partial sequencing of IS1089 revealed similarity of this element with IS1071 of the chlorobenzoate catabolic transposon Tn5271 of Alcaligenes sp. BR60.


Intracranial aneurysms: detection and characterization with MR angiography with use of an advanced postprocessing technique in a blinded-reader study.
To assess magnetic resonance (MR) angiography for the detection and characterization of angiographically proved intracranial aneurysms by using an advanced method of postprocessing, in a blinded-reader study. One hundred fifty-eight vessels were examined with catheter angiography and three-dimensional time-of-flight MR angiography in 44 patients with 63 aneurysms and 15 patients with no aneurysm at catheter angiography. Postprocessing was performed off-line with an advanced multifeature-extraction, ray-tracing algorithm. MR angiograms were interpreted independently by three neuroradiologists blinded to the catheter angiographic results for presence, location, size, and morphology of the aneurysm. Proof of diagnosis was consensus reading of catheter angiograms. Mean sensitivity for detection of aneurysms was 75% (range, 70%-79%). As a screening tool (ie, detection of at least one aneurysm necessitating catheter angiography), mean sensitivity was 91% for all aneurysms and 95% for aneurysms larger than 3 mm. This method was not adequate for detection of lobulation or size of aneurysm. MR angiography with an advanced method of postprocessing can result in highly sensitive, specific studies for the diagnosis of intracranial aneurysms that are of sufficient size to be considered for surgical treatment, but it is inadequate for characterization of aneurysms.


A systematic review of comorbidities and outcomes of adult patients with pleural infection.
Pleural infection remains an important cause of mortality. This study aimed to investigate worldwide patterns of pre-existing comorbidities and clinical outcomes of patients with pleural infection. Studies reporting on adults with pleural infection between 2000 and 2017 were identified from a search of Embase and MEDLINE. Articles reporting exclusively on tuberculous, fungal or post-pneumonectomy infection were excluded. Two reviewers assessed 20 980 records for eligibility. 211 studies met the inclusion criteria. 134 articles (227 898 patients, mean age 52.8 years) reported comorbidity and/or outcome data. The majority of studies were retrospective observational cohorts (n=104, 78%) and the most common region of reporting was East Asia (n=33, 24%) followed by North America (n=27, 20%). 85 articles (50 756 patients) reported comorbidity. The median (interquartile range (IQR)) percentage prevalence of any comorbidity was 72% (58-83%), with respiratory illness (20%, 16-32%) and cardiac illness (19%, 15-27%) most commonly reported. 125 papers (192 298 patients) reported outcome data. The median (IQR) length of stay was 19 days (13-27 days) and median in-hospital or 30-day mortality was 4% (IQR 1-11%). In regions with high-income economies (n=100, 74%) patients were older (mean 56.5 versus 42.5 years, p<0.0001), but there were no significant differences in prevalence of pre-existing comorbidity nor in length of hospital stay or mortality. Patients with pleural infection have high levels of comorbidity and long hospital stays. Most reported data are from high-income economy settings. Data from lower-income regions is needed to better understand regional trends and enable optimal resource provision going forward.


Isolation of single mammalian cells from adherent cultures by fluidic force microscopy.
The physical separation of individual cells from cell populations for single-cell analysis and proliferation is of wide interest in biology and medicine. Today, single-cell isolation is routinely applied to non-adherent cells, though its application to cells grown on a substrate remains challenging. In this report, a versatile approach for isolating single HeLa cells directly from their culture dish is presented. Fluidic force microscopy is first used to detach the targeted cell(s) via the tunable delivery of trypsin, thereby achieving cellular detachment with single-cell resolution. The cell is then trapped by the microfluidic probe via gentle aspiration, displaced with micrometric precision and either transferred onto a new substrate or deposited into a microwell. An optimised non-fouling coating ensures fully reversible cell capture and the potential for serial isolation of multiple cells with 100% successful transfer rate (n = 130) and a survival rate of greater than 95%. By providing an efficient means for isolating targeted adherent cells, the described approach offers exciting possibilities for biomedical research.


MicroRNA transcriptome profiling in cardiac tissue of hypertrophic cardiomyopathy patients with MYBPC3 mutations.
Hypertrophic cardiomyopathy (HCM) is predominantly caused by mutations in genes encoding sarcomeric proteins. One of the most frequent affected genes is MYBPC3, which encodes the thick filament protein cardiac myosin binding protein C. Despite the prevalence of HCM, disease pathology and clinical outcome of sarcomeric mutations are largely unknown. We hypothesized that microRNAs (miRNAs) could play a role in the disease process. To determine which miRNAs were changed in expression, miRNA arrays were performed on heart tissue from HCM patients with a MYBPC3 mutation (n=6) and compared with hearts of non-failing donors (n=6). 532 out of 664 analyzed miRNAs were expressed in at least one heart sample. 13 miRNAs were differentially expressed in HCM compared with donors (at p<0.01, fold change ≥ 2). The genomic context of these differentially expressed miRNAs revealed that miR-204 (fold change 2.4 in HCM vs. donor) was located in an intron of the TRPM3 gene, encoding an aspecific cation channel involved in calcium entry. RT-PCR analysis revealed a trend towards TRPM3 upregulation in HCM compared with donor myocardium (fold change 2.3, p=0.078). In silico identification of mRNA targets of differentially expressed miRNAs showed a large proportion of genes involved in cardiac hypertrophy and cardiac beta-adrenergic receptor signaling and we showed reduced phosphorylation of cardiac troponin I in the HCM myocardium when compared with donor. HCM patients with MYBPC3 mutations have a specific miRNA expression profile. Downstream mRNA targets reveal possible involvement in cardiac signaling pathways.


Linking hydraulic conductivity and photosynthesis to water-source partitioning in trees versus seedlings.
The relationships between hydraulic and photosynthetic properties in plants have been widely studied, but much less is known about how these properties are linked to water-source partitioning, the spatial and temporal separation of water sources in ecosystems. Plant water-source partitioning is often influenced by the proximity of groundwater from the natural surface. We studied the water acquisition strategy and hydraulic and photosynthetic properties of Tuart (Eucalyptus gomphocephala D.C.), a large coastal tree species that occupies seasonally dry habitats underlain by superficial aquifers. Our goal was to quantify water-source partitioning as the proportion of xylem water derived from the vadose and saturated zones with respect to stage of development and proximity of groundwater. We then sought to associate the proportional contribution of a given water source with xylem hydraulic and photosynthetic properties, thus conferring a linkage. Seedlings were more inclined to use surface soil water when rainfall recharge of the upper profile occurred, suggesting that they maintained or rapidly developed a proportionally high amount of functional roots in the upper, seasonally dry, soil profile. This strategy was associated with a lower xylem-area-specific hydraulic conductivity (K(S)), leaf-area-specific hydraulic conductivity (K(L)) and maximum photon yield of photosystem II (F(V)/F(M)). In contrast, trees acquired water from a variety of sources in different seasons and had a higher K(S), K(L) and F(V)/F(M). Despite the higher K(S) and K(L) in trees, the midday hydrodynamic water potential gradient from soil to leaves, ΔΨ, was similar. We conclude that there was a linkage between hydraulic and photosynthetic properties with the partitioning of water sources and that this adaptation to long-term hydrological regimes accommodated the different hydraulic characteristics and hydrological environments of trees versus seedlings.


Impact of a winter respiratory virus season on patients with COPD and association with influenza vaccination.
We assessed the effects of an influenza season on patients with COPD. Data from 2,215 veterans in a multicenter, randomized, double-blind influenza vaccine efficacy study were analyzed for changes in spirometric and functional status, comparing patients with laboratory-documented influenza (LDI)-caused illness, non-LDI-caused respiratory illness, or no illness, and for association with influenza vaccination. Patients received either IM trivalent inactivated influenza virus vaccine (TIV) plus intranasal trivalent, live attenuated, cold-adapted influenza virus vaccine (TC) or TIV plus intranasal placebo (TP). We performed spirometry, measured the chronic lung disease severity index (CLDSI) score to assess functional status and well-being, and tested for influenza virus infection. Worsening in FEV(1), percentage of predicted FEV(1), and CLDSI score (p < 0.001) was associated with acute respiratory illness in 585 illnesses including 94 LDI-caused illnesses. LDI-caused illness was more likely to be associated with worsening in FEV(1) and CLDSI score acutely than non-LDI-caused illness (p < 0.01). Logistic regression showed acute respiratory illness (odds ratio [OR], 1.78; 95% confidence limit [CL], 1.40 to 2.26) to be associated with worsening in CLDSI score, and receipt of TC (OR, 1.39; 95% CL, 1.10 to 1.74) and no illness (OR, 0.70; 95% CL, 0.53 to 0.91 for acute respiratory illness) to be associated with better CLDSI score at the end of the study. Hospitalization was more frequent in patients with acute respiratory illness (p < 0.0001). Acute respiratory illness was associated with increased health-care utilization and obstruction to airflow, and worse functional status and well-being. At the end of the study, receipt of TC was associated with improvement and acute respiratory illness was associated with worsening in functional status and well-being.


Differential evolution of duplicated medakafish mitf genes.
Gene duplication is a major force of evolution. One whole genome duplication (WGD) event in the fish ancestor generated genome-wide duplicates in all modern species. Coloration and patterning on the animal body surface exhibit enormous diversity, representing a mysterious and ideal system for understanding gene evolution. Surface colors and patterns are determined primarily by pigment cells in the skin and eye. Thus, microphthalmia-associated transcription factor (Mitf) as a master regulator of melanocyte development is excellent for studying the evolution of WGD-derived gene duplicates. Here we report the evolution of mitf duplicate, mitf1 and mitf2, in the fish medaka (Oryzias latipes), which encode medaka co-homologs Mitf1 and Mitf2 of the mouse Mitf. Compared to mitf1, mitf2 exhibits an accelerated sequence divergence and loses melanocytic expression in embryos at critical developmental stages. Compared to a Xiphophorus counterpart, the medaka Mitf2 displayed a reduced activity in activating melanogenic gene expression by reporter assays and RT-PCR analyses. We show that the medaka Mitf2 has the ability to induce melanocyte differentiation in medaka embryonic stem cells but at a remarkably reduced efficiency compared to the Xiphophorus counterpart. Our data suggest differential evolution of the medaka mitf duplicate, with mitf1 adopting conservation and mitf2 employing degeneration, which is different from the duplication-degeneration-complementation proposed as the mechanism to preserve many gene duplicates in zebrafish. Our finding reveals species-specific variations for mitf duplicate evolution, in agreement with enormous diversity of body coloration and patterning.


Expression and function of survivin in canine osteosarcoma.
Osteosarcoma has a high mortality rate and remains in need of more effective therapeutic approaches. Survivin is an inhibitor of apoptosis family member protein that blocks apoptosis and drives proliferation in human cancer cells where it is commonly elevated. In this study, we illustrate the superiority of a canine osteosarcoma model as a translational tool for evaluating survivin-directed therapies, owing to the striking similarities in gross and microscopic appearance, biologic behavior, gene expression, and signaling pathway alterations. Elevated survivin expression in primary canine osteosarcoma tissue correlated with increased histologic grade and mitotic index and a decreased disease-free interval (DFI). Survivin attenuation in canine osteosarcoma cells inhibited cell-cycle progression, increased apoptosis, mitotic arrest, and chemosensitivity, and cooperated with chemotherapy to significantly improve in vivo tumor control. Our findings illustrate the utility of a canine system to more accurately model human osteosarcoma and strongly suggest that survivin-directed therapies might be highly effective in its treatment.


Change of treatment modalities over the last 10 years in pediatric patients with inflammatory bowel disease in Switzerland.
During the past decade, several new drugs were approved for the treatment of pediatric inflammatory bowel disease (IBD). We aimed to evaluate if and how pharmacologic treatment options for pediatric IBD in Switzerland have changed over time. Data from the pediatric Swiss IBD Cohort Study, a national prospective cohort study initiated in 2006, were analyzed. Patients were divided into two groups: patients with IBD diagnosis until 2009 (168 patients) and patients with IBD diagnosis in 2010 and after (210 patients). Both groups were analyzed regarding the past and the current therapies as well as need for surgery. Overall, 378 pediatric patients with IBD were analyzed, of which 51.9% had Crohn's disease (CD) and 48.1% had ulcerative colitis/indeterminate colitis. Median age at diagnosis was 12 years. The majority (65.4%) of the patients with ulcerative colitis experienced pancolitis, whereas 45.4% of patients with CD presented with ileocolonic disease at diagnosis. A decreased use of corticosteroids in pediatric patients with CD can be found after 2010 (P=0.041). Use of 5-aminosalicylic acid for patients with CD was dramatically reduced after the year 2010 (33.5 vs. 67.7% after 6 years of disease). A significant shift toward earlier use of biologicals could be shown after 2010 (P<0.001). However, there was no significant decrease of surgery rate after 5 years of disease. In the past decade, a significant earlier use of anti-tumor necrosis factor-α agents in pediatric patients with IBD was observed with steroid-sparing effect in patients with CD. However, this change was not associated with reduction of surgery.


Initiation of growth arrest and apoptosis of MCF-7 mammary carcinoma cells by tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate, is associated with mitochondrial activity.
We investigated the effects of tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate and an approved food additive, establishing induction of growth arrest and apoptosis of MCF-7 human mammary carcinoma cells. Transient increased mitochondria-associated bax, dissipation of the mitochondrial membrane potential (delta(psi)m), and caspase-3-independent cleavage of poly(ADP-ribose) polymerase are evident as early as 4 h after treatment of cells with tributyrin. These events are followed by the transient accumulation of mitochondrial cytochrome c in the cytosol and, finally, the generation and accumulation of cells with subdiploid DNA content. During the period in which mitochondria-associated bax levels are elevated, the delta(psi)m is disrupted, and cytochrome c is detected in the cytosol, we show induction of p21WAF1/Cip1 in the absence of increased p53 and arrest of cells in G2-M. Thus, early mitochondria-associated events may play a key role in initiating and/or coordinating tributyrin-mediated growth arrest and apoptosis of wild-type p53 MCF-7 cells. Because effective chemoprevention has been associated with agents that restore or maintain the balance between proliferation and apoptosis, dietary tributyrin, particularly during the critical period of mammary gland development, may be a promising chemopreventive agent.


Naked RNA immunization with replicons derived from poliovirus and Semliki Forest virus genomes for the generation of a cytotoxic T cell response against the influenza A virus nucleoprotein.
The potential of RNA-based vaccines was evaluated for the generation of a protective immune response in the mouse model of influenza type A virus infection using the internal nucleoprotein (NP) as antigen. This antigen is of particular interest, since it has the potential to elicit protective cytotoxic T lymphocytes (CTL) against heterologous strains of influenza A virus. In view of the short half-life of RNA, self-replicating RNAs or replicons of the positive-stranded genomes of Semliki Forest virus (SFV) and poliovirus were engineered to synthesize the influenza A virus NP in place of their structural proteins. NP expression was demonstrated by immunoprecipitation after transfection of cells with RNA from the SFV (rSFV-NP) and poliovirus (rDeltaP1-E-NP) genome-derived replicons transcribed in vitro. C57BL/6 mice were injected intramuscularly with these synthetic RNAs in naked form. Both replicons, rSFV-NP and rDeltaP1-E-NP, induced antibodies against the influenza virus NP, but only mice immunized with the rSFV-NP replicon developed a CTL response against the immunodominant H-2D(b) epitope NP366. Finally, the protective potential of the CTL response induced by immunization of mice with rSFV-NP RNA was demonstrated by the reduction of virus load in the lungs after challenge infection with mouse-adapted influenza A/PR/8/34 virus and was comparable to the protective potential of the response induced by plasmid DNA immunization. These results demonstrate that naked RNA immunization with self-replicating molecules can effectively induce both humoral and cellular immune responses and constitutes an alternative strategy to DNA immunization.


The late endosome is essential for mTORC1 signaling.
The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein synthesis, cell growth, and autophagy. To examine how endocytic trafficking might be involved in nutrient regulation of mTORC1, we perturbed specific endocytic trafficking pathways and measured mTORC1 activity using S6K1 as a readout. When early/late endosomal conversion was blocked by either overexpression of constitutively active Rab5 (Rab5CA) or knockdown of the Rab7 GEF hVps39, insulin- and amino acid-stimulated mTORC1/S6K1 activation were inhibited, and mTOR localized to hybrid early/late endosomes. Inhibition of other stages of endocytic trafficking had no effect on mTORC1. Overexpression of Rheb, which activates mTOR independently of mTOR localization, rescued mTORC1 signaling in cells expressing Rab5CA, whereas hyperactivation of endogenous Rheb in TSC2-/- MEFs did not. These data suggest that integrity of late endosomes is essential for amino acid- and insulin-stimulated mTORC1 signaling and that blocking the early/late endosomal conversion prevents mTOR from interacting with Rheb in the late endosomal compartment.


Report - Renovation of a traditional Ergh-al-Nassa pill (Hab) to a standard Pharmaceutical molded tablet.
The Ergh-al-Nassa pill (Hab) is a traditional combination suggested as one of the most effective preparations useful for treatment of sciatica. Although traditional preparations can be applied as new therapeutic drugs for investigations and clinical trials, they need to be reformulated to achieve pharmacopoeial standards for modern medicine. In this research, based on seven traditional Persian pharmacopeias for Ergh-al-NassaHab, nine different molded tablets were reformulated. Each formulation comprised the same amount of colchicum, ginger, aloe and yellow myrobalan fruit. Sweet almond oil had to be added in the maximum amount needed to be absorbed by the yellow myrobalan fruit according to its particle size (30-40 mesh sizes). The studies were performed in order to optimize the formulation process according to the role of three levels in particle size of the herbal ingredients (60-70, 80-100, 100-150 mesh sizes) and three levels of initial water for granulation. The molded tablets were evaluated according to standard quality controls for tablets (mass uniformity, LOD, hardness, friability, and disintegration time at 20 and 30 min). Myrobalan powdered to 30-40 mesh size absorbed the maximum amount of sweet almond oil (1:0.75 w/v). The best formulations occurred when the particle size of colchicum, ginger, and aloe was 60-70 mesh size with an initial moisture content of 0.47 ml per 1g of dried powder. The outcome of this research is a pharmaceutical standardized formulation from the traditional Ergh-al-Nassa pill which can be suggested as a sample drug discovery based on traditional knowledge.


Stereotactic radiosurgery for cerebellopontine angle meningiomas.
To assess the long-term outcomes of stereotactic radiosurgery (SRS) for cerebellopontine angle (CPA) meningiomas, the authors retrospectively reviewed data from a 20-year experience. They evaluated progression-free survival as well as improvement, stabilization, or deterioration in clinical symptoms. Seventy-four patients with CPA meningiomas underwent SRS involving various Gamma Knife technologies between 1990 and 2010. The most common presenting symptoms were dizziness or disequilibrium, hearing loss, facial sensory dysfunction, and headache. The median tumor volume was 3.0 cm(3) (range 0.3-17.1 cm(3)), and the median radiation dose to the tumor margin was 13 Gy (range 11-16 Gy). The median follow-up period was 40 months (range 4-147 months). At last imaging follow-up, the tumor volume had decreased in 46 patients (62%), remained stable in 26 patients (35%), and increased in 2 patients (3%). The progression-free survival after SRS was 98% at 1 year, 98% at 3 years, and 95% at 5 years. At the last clinical follow-up, 23 patients (31%) showed neurological improvement, 43 patients (58%) showed no change in symptoms or signs, and 8 patients (11%) had worsening symptoms or signs. The neurological improvement rate after SRS was 16% at 1 year, 31% at 3 years, and 40% at 5 years. The post-SRS deterioration rate was 5% at 1 year, 10% at 3 years, and 16% at 5 years. A multivariate analysis demonstrated that trigeminal neuralgia was the symptom most likely to worsen after SRS (HR 0.08, 95% CI 0.02-0.31; p = 0.001). Asymptomatic peritumoral edema occurred in 4 patients (5%) after SRS, and symptomatic adverse radiation effects developed in 7 patients (9%). Stereotactic radiosurgery for CPA meningiomas provided a high tumor control rate and relatively low risk of ARE. Tumor compression of the trigeminal nerve by a CPA meningioma resulted in an increased rate of facial pain worsening in this patient experience.


Meta-Analysis of Randomized Controlled Trials of Acupuncture for Cancer-Related Fatigue.
Fatigue is a distressing and pervasive problem for people with cancer. In recent years, acupuncture has gained increasing attention among researchers as an alternative management strategy for cancer-related fatigue (CRF). This review aimed to evaluate the effectiveness of acupuncture for CRF. Five databases (Medline, CINAHL, Scopus, the Cochrane Library, and CAJ Full-text Database) were searched up to May 2013. Randomized controlled trials (RCTs) of acupuncture for the treatment of CRF were considered for inclusion. Seven RCTs were included for meta-analysis, involving a total of 689 subjects. Three studies compared acupuncture with sham acupuncture for CRF with follow-up at 10 weeks; the standardized mean difference (SMD) for general CRF change values was -0.82 (95% confidence interval [CI] = -1.90 to 0.26). When acupuncture plus education intervention was compared with usual care, there was a statistically significant difference for the change score of general CRF (SMD = -2.12; 95% CI = -3.21 to -1.03). The SMD for general CRF change scores between acupuncture with no treatment or wait-list control was -1.46 (95% CI = -3.56 to 0.63). Finally, the SMD for general CRF change scores between acupuncture with acupressure or self-acupuncture was -1.12 (95% CI = -3.03 to 0.78). Three trials reported data for general quality of life and functioning status, reporting enough data for statistical pooling but showing no statistically significant difference (Zscore = 1.15,P= .25, SMD = 0.99, 95% CI = -0.70 to 2.68 andZscore = 1.13,P= .26, SMD = 1.38, 95% CI = -1.02 to 3.79, respectively). TheI(2)statistics of all statistically pooled data were higher than 50%, indicating heterogeneity between the trials. There were 4 sets of comparison for the effectiveness of acupuncture for CRF; statistical pooling of the reduction in CRF from baseline to follow-up showed in favor of acupuncture. However, 3 sets of comparison for the pooled estimates of effect sizes had no statistical significance. Although one set of comparison (acupuncture plus education interventions vs usual care) had statistically significant differences, it is unclear whether this pooled positive outcome is attributable to the effects of acupuncture or to the education intervention. In addition, the duration of follow-up in these included trials was up to 10 weeks, and some RCTs had methodological flaws. Further rigorously designed RCTs adhering to acceptable standards of trial methodology are required to determine the effectiveness of acupuncture and its long-term effects on CRF.


The modeling and analysis of transesterification reaction conditions in the selection of optimal biodiesel yield and viscosity.
Among alternative fuels, biodiesel has been emphasized as a substantial candidate for diesel engines because of many advantages. However, the main shortcomings preventing more widespread use of biodiesel are high production cost and viscosity. In order to simultaneously overcome both of these shortcomings, the reaction conditions for the transesterification of waste cooking oil (WCO) were optimized using Taguchi and the full factorial design approaches. The analyses of signal to noise ratio and variance were also performed to identify the dominance of reaction conditions on viscosity and biodiesel yield. As a result, the optimal reaction conditions giving the lowest kinematic viscosity (3.991 cSt) and the highest biodiesel yield (98.19%) were determined to be as follows: sodium methoxide amount of 1.00 wt%, reaction time of 60 min, reaction temperature of 55 °C, and methanol to oil molar ratio of 6:1. The catalyst amount and methanol to oil molar ratio were found to be the most significant conditions influencing on the viscosity (10.36% and 78.87% contributions) and the yield (58.48% and 20.17% contributions), respectively. Finally, all physicochemical properties of final waste cooking oil biodiesel (WCOB) produced under optimal reaction conditions were found to meet the EN 14214.