Etiology of chronic low back pain in patients having undergone lumbar fusion.
To estimate the prevalence of lumbar internal disc disruption, zygapohyseal joint pain, sacroiliac joint pain, and soft tissue irritation by fusion hardware in post-fusion low back pain patients compared with non-fused patients utilizing diagnostic spinal procedures. Retrospective chart review. University spine center. Patients presenting to a community-based, multidisciplinary, academic spine center (65.9% female, mean age 54.4 years, median pain duration 12 months). Charts of consecutive low back pain cases completing diagnostic spinal procedures including provocation discography and zygapohyseal joint, sacroiliac joint, and fusion hardware blockade were retrospectively reviewed. Based on the results of discography and/or diagnostic blockades, subjects were classified with internal disc disruption, zygapohyseal joint pain, sacroiliac joint pain, or fusion hardware related pain. The diagnoses of 28 fusion cases identified from 170 low back pain patients undergoing diagnostic procedures included 12 with sacroiliac joint pain, seven with internal disc disruption, five with zygapohyseal joint pain, and four due to soft tissue irritation from fusion hardware. No significant differences were noted in zygapohyseal joint mediated pain with and without fusion history. Mean ages of patients were similar with and without fusion history for cases diagnosed as internal disc disruption. In patients' recalcitrant to non-interventional care, the sacroiliac joint is the most likely source of low back pain after lumbar fusion followed by internal disc disruption, zygapohyseal joint pain, and soft tissue irritation due to fusion hardware. Sacroiliac joint pain is more common after fusion, while internal disc disruption is more common in non-fusion patients.


Encapsulation cell therapy for mucopolysaccharidosis type VII using genetically engineered immortalized human amniotic epithelial cells.
Mucopolysaccharidosis type VII (MPSVII) is a lysosomal storage disease resulted from a deficiency of the enzyme beta-glucuronidase (GUSB), which is necessary for degradation of glycosaminoglycans (GAGs). The deficiency of GUSB causes progressive accumulation of GAGs and subsequent lysosomal distension in multiple tissues, including the central nervous system (CNS). In murine experiments, bone marrow transplant, enzyme replacement, viral vectors, and genetically modified cells were successfully used for correction of the visceral accumulation of GAGs, but little improvement was seen in the brain, because these therapeutic agents cannot cross the blood-brain barrier (BBB). Although direct intracerebral injection of GUSB-encoding viral vectors has been developed to bypass the BBB, the possibility of tumor formation and the toxicity of over-expressed GUSB have been reported. In this study, we generated immortalized human amniotic epithelial (IHAE) cells to maintain the effect of implantation, and encapsulated these cells to prevent harmful immunological response and tumor formation and to regulate the level of GUSB expression within the host. Moreover, we generated IHAE cells that over-express and secrete human GUSB following transduction with an adenoviral vector encoding human GUSB. Therapeutic efficacy for MPSVII was evaluated in and ex vivo experiments using these encapsulated genetically engineered GUSB-encoding IHAE cells. We confirmed that encapsulated genetically engineered IHAE cells could secrete significant amounts of GUSB outside the capsule in vitro and into the cerebral parenchyma of C3H mice seven days after the capsule implantation. Thus, encapsulation cell therapy using genetically engineered IHAE cells is an effective armamentarium for the treatment of MPSVII.


Control of laser heating in clusters through variation in temporal pulse shape.
We present results of experiments and simulations of the interaction between a high intensity, femtosecond laser pulse and an atomic cluster that show the temporal profile of the laser pulse plays a crucial role in the expansion dynamics of the cluster. Experiments were conducted in rare gas clusters of Xe, Kr and Ar of radius 30 - 80 A with a laser pulse of duration 70 - 240 fs and a peak intensity of ~1016 Wcm-2. The shape of the laser pulse was a Gaussian with a shoulder of intensity 0.02 times the peak pulse intensity appearing on either the rising or falling edge of the main pulse depending on the sign of chirp applied to the laser pulse. Significant differences (up to a factor of 2) in the energies of the ions obtained from the laser-cluster interaction were measured when the shape of the laser pulse was varied.


SU-E-T-15: Small and Nonstandard Photon Field Dosimetry Characterization Using Monte Carlo Methods.
To quantify uncertainty reduction in small photon field dosimetry through characterization of ionization chambers and calibration conditions using detailed Monte Carlo methods benchmarked against NIST-traceable measurements. Phase space profiles were obtained using detailed EGSnrc Monte Carlo models for a Varian 6 MV photon linear accelerator, and a NIST-traceable cobalt-60 teletherapy unit. The responses of a farmer-type ionization chamber, two micro-ionization chambers, and one scanning-type ionization chamber were simulated in multiple calibration conditions. Calibration conditions included static field sizes ranging from (10×10) cm squared to (0.5×0.5) cm squared for the 6 MV and cobalt-60 beam qualities and an additional dynamic IMRT plan for the 6 MV beam quality. Calibration conditions also consisted of ionization chambers placed in a standard water phantom and in a specially designed acrylic phantom. Tolerance limits on the calibration conditions were investigated. All models were benchmarked against measured beam quality data, including ionization chamber beam quality correction factors for the standard absorbed dose to water cobalt-60 calibration coefficient. The majority of the simulated small field response values fell within the uncertainty of the measured values. A database was created for several proposed small field calibration conditions to provide comparisons with the Co-60 standard reference conditions. The database includes the small field calibration conditions' beam quality correction factors, tolerances, and dose calibration uncertainties. The characterization of multiple calibration conditions provided an improved understanding of how the cobalt-60 ionization chamber absorbed dose to water calibration coefficient from an ADCL can optimally be applied to small and nonstandard field calibrations to reduce the associated dose uncertainty. The developed methodology will contribute to future research of other small field radiotherapy modalities. The resulting database provides support for future recommendations on the implementation of small and non-standard field calibration protocols.


Hypertension in African Populations: Review and Computational Insights.
Hypertension (HTN) is a persistent public health problem affecting approximately 1.3 billion individuals globally. Treatment-resistant hypertension (TRH) is defined as high blood pressure (BP) in a hypertensive patient that remains above goal despite use of ≥3 antihypertensive agents of different classes including a diuretic. Despite a plethora of treatment options available, only 31.0% of individuals have their HTN controlled. Interindividual genetic variability to drug response might explain this disappointing outcome because of genetic polymorphisms. Additionally, the poor knowledge of pathophysiological mechanisms underlying hypertensive disease and the long-term interaction of antihypertensive drugs with blood pressure control mechanisms further aggravates the problem. Furthermore, in Africa, there is a paucity of pharmacogenomic data on the treatment of resistant hypertension. Therefore, identification of genetic signals having the potential to predict the response of a drug for a given individual in an African population has been the subject of intensive investigation. In this review, we aim to systematically extract and discuss African evidence on the genetic variation, and pharmacogenomics towards the treatment of HTN. Furthermore, in silico methods are utilized to elucidate biological processes that will aid in identifying novel drug targets for the treatment of resistant hypertension in an African population. To provide an expanded view of genetic variants associated with the development of HTN, this study was performed using publicly available databases such as PubMed, Scopus, Web of Science, African Journal Online, PharmGKB searching for relevant papers between 1984 and 2020. A total of 2784 articles were reviewed, and only 42 studies were included following the inclusion criteria. Twenty studies reported associations with HTN and genes such as AGT (rs699), ACE (rs1799752), NOS3 (rs1799983), MTHFR (rs1801133), AGTR1 (rs5186), while twenty-two studies did not show any association within the African population. Thereafter, an in silico predictive approach was utilized to identify several genes including CLCNKB, CYPB11B2, SH2B2, STK9, and TBX5 which may act as potential drug targets because they are involved in pathways known to influence blood pressure. Next, co-expressed genes were identified as they are controlled by the same transcriptional regulatory program and may potentially be more effective as multiple drug targets in the treatment regimens for HTN. Genes belonging to the co-expressed gene cluster, ACE, AGT, AGTR1, AGTR2, and NOS3 as well as CSK and ADRG1 showed enrichment of G-protein-coupled receptor activity, the classical targets of drug discovery, which mediate cellular signaling processes. The latter is of importance, as the targeting of co-regulatory gene clusters will allow for the development of more effective HTN drug targets that could decrease the prevalence of both controlled and TRH.


Long-Term Response to Sunitinib Treatment in Metastatic Renal Cell Carcinoma: A Pooled Analysis of Clinical Trials.
We characterized clinical outcomes of patients with metastatic renal cell carcinoma (mRCC) treated with sunitinib who were long-term responders (LTRs), defined as patients having progression-free survival (PFS) > 18 months. A retrospective analysis of data from 5714 patients with mRCC treated with sunitinib in 8 phase II/III clinical trials and the expanded access program. Duration on-study and objective response rate (ORR) were compared between LTRs and patients with PFS ≤ 18 months ("others"). PFS and overall survival (OS) were summarized using Kaplan-Meier methodology. Overall, 898 (15.7%) patients achieved a long-term response and 4816 (84.3%) patients did not achieve long-term response. The median (range) duration on-study was 28.6 (16.8-70.7) months in LTRs and 5.5 (0-68.8) months in others. ORR was 51% in LTRs versus 14% in others (P < .0001). Median PFS in LTRs was 32.11 months and median OS was not reached. LTRs had higher percentage of early tumor shrinkage ≥ 10% at the first scan (67.1% vs. 51.2%; P = .0018) and greater median maximum on-study tumor shrinkage from baseline (-56.9 vs. -27.1; P < .0001) versus others. White race, Eastern Cooperative Oncology Group performance status 0, time from diagnosis to treatment ≥ 1 year, clear cell histology, no liver metastasis, lactate dehydrogenase ≤ 1.5 upper limit of normal (ULN), corrected calcium ≤ 10 mg/dL, hemoglobin greater than the lower limit of normal, platelets less than or equal to ULN, body mass index ≥ 25 kg/m2, and low neutrophil-to-lymphocyte ratio were associated with LTR. A subset of patients with mRCC treated with sunitinib achieved long-term response. LTRs had improved ORR, PFS, and OS.


Gsx2 is required for specification of neurons in the inferior olivary nuclei from Ptf1a-expressing neural progenitors in zebrafish.
Neurons in the inferior olivary nuclei (IO neurons) send climbing fibers to Purkinje cells to elicit functions of the cerebellum. IO neurons and Purkinje cells are derived from neural progenitors expressing the proneural gene ptf1a In this study, we found that the homeobox gene gsx2 was co-expressed with ptf1a in IO progenitors in zebrafish. Both gsx2 and ptf1a zebrafish mutants showed a strong reduction or loss of IO neurons. The expression of ptf1a was not affected in gsx2 mutants, and vice versa. In IO progenitors, the ptf1a mutation increased apoptosis whereas the gsx2 mutation did not, suggesting that ptf1a and gsx2 are regulated independently of each other and have distinct roles. The fibroblast growth factors (Fgf) 3 and 8a, and retinoic acid signals negatively and positively, respectively, regulated gsx2 expression and thereby the development of IO neurons. mafba and Hox genes are at least partly involved in the Fgf- and retinoic acid-dependent regulation of IO neuronal development. Our results indicate that gsx2 mediates the rostro-caudal positional signals to specify the identity of IO neurons from ptf1a-expressing neural progenitors.


Bionomics of Culex quinquefasciatus within urban areas of Rio de Janeiro, Southeastern Brazil.
To evaluate density, parity rates, daily survival and longevity of natural populations of Culex quinquefasciatus in three neighborhoods with distinct socio-economic and infrastructure profiles. Mosquito collections of the Culex quinquefasciatus species were performed weekly during two four month periods, from August to November 2008 (spring) and March to June 2009 (fall), in a favela (slum), a suburban area and a middle class area of Rio de Janeiro, Southeastern Brazil. Collections were performed with backpack aspirators, in 20 randomly selected houses in each area per week, during 15-20 minutes per house. Ovaries were removed from captured females and classified as initial, intermediary or final stage. Furthermore, females were dissected for determination of parity based on the condition of the tracheal system. Mosquito survival rate and longevity were estimated on a per month basis for each neighborhood. We collected a total of 2,062 Culex quinquefasciatus, but monthly vector density was not correlated with temperature and rainfall. We dissected the ovaries of 625 Culex quinquefasciatus, and overall, there was a higher proportion of nulliparous females during the dryer months, while gravid females were more frequent in rainy months. In the middle class neighborhood, the parity rate reached up to 93.75% with survivorship of 0.979. Lower parity and survival rates were obtained in the suburban area (as low as 36.4% parity and 0.711 daily survival). Up to 84.7% of Culex quinquefasciatus females could survive the eight day period needed to complete West Nile Virus incubation. The survival rate of Culex quinquefasciatus varied significantly between the neighborhoods. This suggests that vectorial capacity and disease transmission risk may vary greatly between different urban areas, which is potentially useful information for vector control programs.


Bacterial Stripe of Hog Millet Caused by Acidovorax avenae subsp. avenae, a New Disease in Korea.
In July 2011, bacterial stripe was observed on a commercial field of hog millet (Panicum miliaceum L.) in Chuncheon, Korea, with a disease incidence of 37% in the field. Symptoms on leaves included reddish-brown, long, narrow stripes that varied in length and were sharply delineated by uninfected adjacent vascular bundles. Eleven bacterial isolates (BC3107, BC3214 to BC3223) were recovered on trypticase soy agar from lesions surface sterilized in 70% ethanol for 1 min. The isolates, all obtained from different plants, were gram negative, oxidase positive, aerobic rods with two to four flagella. The isolates produced circular, cream-colored, nonfluorescent, butyrous colonies with entire margins on King's B medium. Using the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), the isolates were identified as Acidovorax avenae subsp. avenae with Biolog similarity indices ranging from 0.52 to 0.72 after 24 hr. Characters for differentiating between Acidovorax spp. were tested according to Schaad et al. (2). The isolates were positive for gelatin liquefaction, nitrate reduction, lipase production, utilization of D-mannitol, sodium citrate, and alkaline in litmus milk. The isolates were negative for utilization of D-arabitol and did not amplify with PCR primer sets Aaaf5, Aaaf3/Aaar2, and Aacf2/Aacr2. Colonies were V-, V+, and V+ for utilization of D-fucose, maltose, and ethanol, respectively. Regions of the 16S rRNA (rrs) and the IGS were sequenced to aid in the identification of the isolates using reported PCR primer sets (1,4). A 1,426 bp fragment of the rrs region shared 100% similarity with all strains of A. avenae available in GenBank. Pathogenicity tests were separately performed for the 11 isolates in different greenhouses located in Suwon (National Academy of Agricultural Science), and Chuncheon (Gangwondo Agricultural Research and Extension Services) in Korea. Pathogenicity was confirmed by clip inoculation with sterilized scissors dipped into cell suspensions containing 105 CFU/ml on three 8-day-old leaves of hog millet (two plants per isolate), rice (Oryza sativa L. cv. Hopyeong), and sweet corn (Zea mays L. cv. Daehak) in a greenhouse maintained at 28 to 32°C and 90% relative humidity. The isolates induced similar symptoms as those originally observed on hog millet 5 days after inoculation. No symptoms were observed on the control plants (hog millet, rice, and sweet corn), which were clipped with scissors dipped in sterilized distilled water. The identity of bacteria reisolated from the stripes on inoculated leaves was confirmed by analyzing sequences of the 16S-23S rRNA intergenic spacer region (IGS) (1). On the basis of physiological, pathological, and sequence data, the isolates were identified as A. avenae subsp. avenae. To our knowledge, this is the first report of bacterial stripe of hog millet caused by A. avenae subsp. avenae in Korea. The spread of the bacterial disease is expected to have a significant economic impact on hog millet culture in the fields of Gangwon Province in Korea. Nucleotide sequence data reported are available under accession numbers JQ743877 to JQ743887 for rrs of BC 3207 and BC3214 to BC3223, and JQ743877 to JQ743887 for IGS of BC3207 and BC3214 to BC3223. References: (1) T. Barry et al. The PCR Methods Appl. 1:51, 1991. (2) N. W. Schaad et al. Syst, Appl. Microbiol. 31: 434, 2008. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) W. G. Weisburg et al. J. Bacteriol. 173: 697, 1991.


The association between serum albumin levels and metabolic syndrome in a rural population of Korea.
A positive association between serum albumin levels and metabolic syndrome has been reported in observation studies, but it has not been established in the Korean population. The purpose of this study was to evaluate the association between serum albumin levels and the presence of metabolic syndrome among a sample of apparently healthy Korean adults. This cross-sectional study analyzed data of 3189 community-dwelling people (1189 men and 2000 women) who were aged 40 to 87 years and were living in a rural area in Korea. Serum albumin levels were classified into quartile groups for each sex. Metabolic syndrome was defined according to the National Cholesterol Education Program Adult Treatment Panel III guidelines with an adjusted waist circumference cut-off value (≥90 cm for men and ≥85 cm for women). An independent association between serum albumin levels and metabolic syndrome was assessed by multiple logistic regression analysis. Higher serum albumin levels were associated with increased prevalence of metabolic syndrome. The odds ratio (95% confidence interval) of the prevalence of metabolic syndrome for the highest versus the lowest serum albumin quartiles was 2.81 (1.91 to 4.14) in men and 1.96 (1.52 to 2.52) in women, after adjusting for age, smoking status, alcohol consumption, and physical activity. When each metabolic abnormality was analyzed separately, higher serum albumin levels were significantly associated with hypertriglyceridemia and hyperglycemia in both sexes, and with abdominal obesity in men. These results suggest that higher serum albumin levels are positively associated with an increased risk of metabolic syndrome in Korean adults.


Inflammatory role of high salt level in tumor microenvironment (Review).
Chronic inflammation is known to play a critical role in cancer development and progression. High salt is known to mediate several chronic inflammatory diseases including hypertension, myocardial infarction, neurological ischemic attack, autoimmune diseases and cancers. High salt level is shown to induce angiogenesis and immune-dysfunction, both of which play a direct role in cancer proliferation. Furthermore, salt has been suggested to enhance Warburg-like metabolic phenotype in cancer cells and at the same time also induce pro-tumor MΦ2-macrophage phenotype. Recent studies have identified several molecular targets such as tonicity specific transcript factor NFAT5/TonEBP, sodium ion channel γENaC, and vascular endothelial growth factor, VEGF, which are upregulated under high salt external environment. These molecular targets offer futuristic therapeutic application in precision medicine. In this review, we discuss the current understanding of the salt mediated metabolic and immune dysfunctions playing a potential role in cancerous changes.


Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions.
Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.


Evaluating the microbial pattern of periprosthetic joint infections of the hip and knee.
Periprosthetic joint infection (PJI) is a devastating complication that leads to enormous economic and health care complaints from affected patients. The aim of this study is to identify the causative pathogens responsible for PJI, evaluate temporal trends concerning the pathogen pattern and identify potential risk factors for PJI. This was a retrospective study analysing a total of 937 patients suffering PJI of the hip or knee joint between 2003 and 2011. In total, 394 patients (42.0 %) with total knee arthroplasty (TKA), 477 patients (50.9 %) with total hip arthroplasty (THA) and 64 patients (6.8 %) receiving a dual-head prosthesis had to be hospitalised due to PJI. In two cases (0.2 %), a simultaneous infection of TKA and THA occurred. The mean age of the study cohort was 70.85±11.68 years. The mean body mass index (BMI) was 28.53±5.7. According to the Charlson comorbidity index, 2.99 % of the patients were classified as severity Grade 1, 13.98 % Grade 2, 40.02 % Grade 3 and 43.0 % Grade 4. Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus epidermidis (MRSE), methicillin-resistant Staphylococcus aureus (MRSA), coagulase-negative Staphylococcus (CoNS), Streptococcus, and Enterococcus were the pathogens mainly responsible. An increase in high-resistance pathogens, such as MRSE, extended-spectrum beta-lactamase bacteria (ESBL), ampicillin-resistant Enterococcus, Acinetobacter spp. and vancomycin-resistant Enterococcus (VRE), was found during the study period. Only MRSA showed a declining tendency in a regression model. Patients suffering PJI present a certain risk profile with many comorbidities, e.g. high age and obesity. The observed microbiological pattern demonstrates the rise of high-resistance pathogens.


A new group of conserved coactivators that increase the specificity of AP-1 transcription factors.
The Jun proteins are nuclear proteins that combine with Fos proteins to form a gene-regulatory protein, AP-1. They have highly conserved DNA-binding and dimerization domains, resulting in almost identical sequence-recognition properties. Nevertheless, there are many indications that each Jun protein activates a distinct and only partially overlapping set of AP-1 target genes. Using the more variable activation domain of c-Jun as a bait, we identified a protein, JAB1, that interacts with c-Jun and JunD, but not with JunB or v-Jun. As a result, JAB1 selectively potentiates transactivation by only c-Jun or JunD. In vitro, JAB1 specifically stabilizes complexes of c-Jun or JunD with AP-1 sites and does not affect binding of either JunB or v-Jun. The amino-terminal half of JAB1 is very similar to the amino terminal region of Pad1 from fission yeast, which was identified genetically as a coactivator of a subset of AP-1 target genes. JAB1 and Pad1 are also functionally interchangeable. They define a new group of coactivators that increase the specificity of target gene activation by AP-1 proteins.


How the medium shapes the message: Printing and the rise of the arts and sciences.
Communication technologies, from printing to social media, affect our historical records by changing the way ideas are spread and recorded. Yet, finding statistical evidence of this fact has been challenging. Here we combine a common causal inference technique (instrumental variable estimation) with a dataset on nearly forty thousand biographies from Wikipedia (Pantheon 2.0), to study the effect of the introduction of printing in European cities on Wikipedia's digital biographical records. By using a city's distance to Mainz as an instrument for the adoption of the movable type press, we show that European cities that adopted printing earlier were more likely to become the birthplace of a famous scientist or artist during the years following the invention of printing. We bring these findings to recent communication technologies by showing that the number of radios and televisions in a country correlates with the number of globally famous performing artists and sports players born in that country, even after controlling for GDP, population, and including country and year fixed effects. These findings support the hypothesis that the introduction of communication technologies can bias historical records in the direction of the content that is best suited for each technology.


A cryptic hydrophobic pocket in the polo-box domain of the polo-like kinase PLK1 regulates substrate recognition and mitotic chromosome segregation.
The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.


Wildfire exposure analysis on the national forests in the Pacific Northwest, USA.
We analyzed wildfire exposure for key social and ecological features on the national forests in Oregon and Washington. The forests contain numerous urban interfaces, old growth forests, recreational sites, and habitat for rare and endangered species. Many of these resources are threatened by wildfire, especially in the east Cascade Mountains fire-prone forests. The study illustrates the application of wildfire simulation for risk assessment where the major threat is from large and rare naturally ignited fires, versus many previous studies that have focused on risk driven by frequent and small fires from anthropogenic ignitions. Wildfire simulation modeling was used to characterize potential wildfire behavior in terms of annual burn probability and flame length. Spatial data on selected social and ecological features were obtained from Forest Service GIS databases and elsewhere. The potential wildfire behavior was then summarized for each spatial location of each resource. The analysis suggested strong spatial variation in both burn probability and conditional flame length for many of the features examined, including biodiversity, urban interfaces, and infrastructure. We propose that the spatial patterns in modeled wildfire behavior could be used to improve existing prioritization of fuel management and wildfire preparedness activities within the Pacific Northwest region.


Biomarkers of early stage osteoarthritis, rheumatoid arthritis and musculoskeletal health.
There is currently no biochemical test for detection of early-stage osteoarthritis (eOA). Tests for early-stage rheumatoid arthritis (eRA) such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibodies require refinement to improve clinical utility. We developed robust mass spectrometric methods to quantify citrullinated protein (CP) and free hydroxyproline in body fluids. We detected CP in the plasma of healthy subjects and surprisingly found that CP was increased in both patients with eOA and eRA whereas anti-CCP antibodies were predominantly present in eRA. A 4-class diagnostic algorithm combining plasma/serum CP, anti-CCP antibody and hydroxyproline applied to a cohort gave specific and sensitive detection and discrimination of eOA, eRA, other non-RA inflammatory joint diseases and good skeletal health. This provides a first-in-class plasma/serum-based biochemical assay for diagnosis and type discrimination of early-stage arthritis to facilitate improved treatment and patient outcomes, exploiting citrullinated protein and related differential autoimmunity.


Effect of biphasic insulin aspart 30 combined with metformin on glycaemic control in obese people with type 2 diabetes.
Combination therapy consisting of biphasic insulin aspart 30 bid with metformin provide better glycaemic control in obese patients with diabetes mellitus type 2. In our study, patients who were treated with 2550 mg of metformin, administered in three daily doses had poor glycaemic control. Three months after switching from metformin therapy to treatment with biphasic insulin aspart 30 + metformin twice a day, glycaemic control improved with significant reduction in hemoglobin HbA1c, fasting blood glucose and postprandial blood glucose levels. Biphasic insulin aspart 30 in combination with metformin administered twice a day may be recommended as a starting insulin treatment in obese diabetic persons whose glycaemic control remained poor while on oral metformin therapy alone.


Tensorial extensions of independent component analysis for multisubject FMRI analysis.
We discuss model-free analysis of multisubject or multisession FMRI data by extending the single-session probabilistic independent component analysis model (PICA; Beckmann and Smith, 2004. IEEE Trans. on Medical Imaging, 23 (2) 137-152) to higher dimensions. This results in a three-way decomposition that represents the different signals and artefacts present in the data in terms of their temporal, spatial, and subject-dependent variations. The technique is derived from and compared with parallel factor analysis (PARAFAC; Harshman and Lundy, 1984. In Research methods for multimode data analysis, chapter 5, pages 122-215. Praeger, New York). Using simulated data as well as data from multisession and multisubject FMRI studies we demonstrate that the tensor PICA approach is able to efficiently and accurately extract signals of interest in the spatial, temporal, and subject/session domain. The final decompositions improve upon PARAFAC results in terms of greater accuracy, reduced interference between the different estimated sources (reduced cross-talk), robustness (against deviations of the data from modeling assumptions and against overfitting), and computational speed. On real FMRI 'activation' data, the tensor PICA approach is able to extract plausible activation maps, time courses, and session/subject modes as well as provide a rich description of additional processes of interest such as image artefacts or secondary activation patterns. The resulting data decomposition gives simple and useful representations of multisubject/multisession FMRI data that can aid the interpretation and optimization of group FMRI studies beyond what can be achieved using model-based analysis techniques.


Evaluation of Clinical Practice Guidelines on Fall Prevention and Management for Older Adults: A Systematic Review.
With the global population aging, falls and fall-related injuries are ubiquitous, and several clinical practice guidelines for falls prevention and management for individuals 60 years or older have been developed. A systematic evaluation of the recommendations and agreement level is lacking. To perform a systematic review of clinical practice guidelines for falls prevention and management for adults 60 years or older in all settings (eg, community, acute care, and nursing homes), evaluate agreement in recommendations, and identify potential gaps. A systematic review following Preferred Reporting Items for Systematic Reviews and Meta-analyses statement methods for clinical practice guidelines on fall prevention and management for older adults was conducted (updated July 1, 2021) using MEDLINE, PubMed, PsycINFO, Embase, CINAHL, the Cochrane Library, PEDro, and Epistemonikos databases. Medical Subject Headings search terms were related to falls, clinical practice guidelines, management and prevention, and older adults, with no restrictions on date, language, or setting for inclusion. Three independent reviewers selected records for full-text examination if they followed evidence- and consensus-based processes and assessed the quality of the guidelines using Appraisal of Guidelines for Research & Evaluation II (AGREE-II) criteria. The strength of the recommendations was evaluated using Grades of Recommendation, Assessment, Development, and Evaluation scores, and agreement across topic areas was assessed using the Fleiss κ statistic. Of 11 414 records identified, 159 were fully reviewed and assessed for eligibility, and 15 were included. All 15 selected guidelines had high-quality AGREE-II total scores (mean [SD], 80.1% [5.6%]), although individual quality domain scores for clinical applicability (mean [SD], 63.4% [11.4%]) and stakeholder (clinicians, patients, or caregivers) involvement (mean [SD], 76.3% [9.0%]) were lower. A total of 198 recommendations covering 16 topic areas in 15 guidelines were identified after screening 4767 abstracts that proceeded to 159 full texts. Most (≥11) guidelines strongly recommended performing risk stratification, assessment tests for gait and balance, fracture and osteoporosis management, multifactorial interventions, medication review, exercise promotion, environment modification, vision and footwear correction, referral to physiotherapy, and cardiovascular interventions. The strengths of the recommendations were inconsistent for vitamin D supplementation, addressing cognitive factors, and falls prevention education. Recommendations on use of hip protectors and digital technology or wearables were often missing. None of the examined guidelines included a patient or caregiver panel in their deliberations. This systematic review found that current clinical practice guidelines on fall prevention and management for older adults showed a high degree of agreement in several areas in which strong recommendations were made, whereas other topic areas did not achieve this level of consensus or coverage. Future guidelines should address clinical applicability of their recommendations and include perspectives of patients and other stakeholders.


The mitochondrial lncRNA ASncmtRNA-2 is induced in aging and replicative senescence in Endothelial Cells.
Age-associated cardiovascular diseases are at least partially ascribable to vascular cell senescence. Replicative senescence (RS) and stress-induced premature senescence (SIPS) are provoked respectively by endogenous (telomere erosion) and exogenous (H2O2, UV) stimuli resulting in cell cycle arrest in G1 and G2 phases. In both scenarios, mitochondria-derived ROS are important players in senescence initiation. We aimed to define whether a mtDNA-transcribed long-non-coding-RNA (lncRNA), ASncmtRNA-2, has a role in vascular aging and senescence. Aortas of old mice, characterized by increased senescence, showed an increment in ASncmtRNA-2 expression. In vitro analysis of Endothelial Cells (EC) and Vascular Smooth Muscle Cells (VSMC) established that ASncmtRNA-2 is induced in EC, but not in VSMC, during RS. Surprisingly, ASncmtRNA-2 is not upregulated in two different EC SIPS scenarios, treated with H2O2 and UV. The p16 gene displayed similar ASncmtRNA-2 expression patterns, suggesting a possible co-regulation of the two genes. Interestingly, the expression of two miRNAs, hsa-miR-4485 and hsa-miR-1973, with perfect homology to the double strand region of ASncmtRNA-2 and originating at least in part from a mitochondrial transcript, was induced in RS, opening to the possibility that this lncRNA functions as a non-canonical precursor of these miRNAs. Cell cycle analysis of EC transiently over-expressing ASncmtRNA-2 revealed an accumulation of EC in the G2/M phase, but not in the G1 phase. We propose that ASncmtRNA-2 in EC might be involved in the RS establishment by participating in the cell cycle arrest in G2/M phase, possibly through the production of hsa-miR-4485 and hsa-miR-1973. This article is part of a Special Issue entitled: Mitochondria.


Balance Deficits and Functional Disability in Cancer Survivors Exposed to Neurotoxic Cancer Treatments.
Chemotherapy-induced peripheral neuropathy (CIPN) persists after treatment in up to 40% of cancer survivors and has been linked with increased balance deficits, disabilities, and fall occurrences. This study aimed to comprehensively assess the links between CIPN, balance deficits, and functional disability and to inform the development of clinical screening tools for patients at risk of these events. A total of 190 cancer survivors exposed to neurotoxic chemotherapies (age, 57 ± 13 years; average time from completion of neurotoxic therapy, 12 ± 11 months) attended a neurology research clinic for a single cross-sectional assessment of patient-reported and objective CIPN, standing balance in 4 conditions of increasing difficulty, and functional disability. Most patients (68%) reported CIPN symptoms at assessment. Symptomatic patients displayed increased functional disability (F=39.4; P<.001) and balance deficits (F=34.5; P<.001), with degree of balance impairments consistent with a healthy elderly population (age ≥65 years) reporting multiple falls over the subsequent year. Increasing CIPN severity correlated with increasing functional disability (clinically assessed R2=0.46; patient-reported R2=0.49; P<.001) and balance deficits (clinically assessed R2=0.41; patient-reported R2=0.30; P<.001). A 5-factor model of key independent correlates-patient-reported numbness/tingling, weakness, and balance deficit; age; and vibration perception-was strongly linked to balance deficits (R2=0.46; P<.001) and functional disability (R2=0.56; P<.001). This study confirms links between increasing CIPN severity and increasing balance deficits and functional disability using comprehensive CIPN assessment methodology. The extent of balance deficits in patients with CIPN underscores the functional consequences of neurotoxicity. A 5-factor model provides a foundation for clinical screening tools to assess balance deficits and functional disability in patients exposed to neurotoxic chemotherapies.


Impact of selected medical conditions on self-reported lower-extremity function in Mexican-American elderly.
To examine the independent impact of common medical conditions on lower-extremity function in Mexican-American elderly. Cross-sectional study using a probability sample of non-institutionalized Mexican Americans aged 65 or older. The five Southwestern states, Texas, New Mexico, Arizona, Colorado and California. All subjects were interviewed in person (n = 2,873) or by proxy (n = 177) in their homes during late 1993 and early 1994. Respondents were asked whether they could perform four activities related to lower-extremity function without help: walking across a small room, getting from a bed to a chair, walking up and down stairs, and walking half a mile. A summary measure of lower body disability created from these four items was regressed on seven common medical conditions plus five control variables using multiple logistic regression. Adjusted Odds Ratios (OR) suggested that impaired lower-extremity function was associated with previous diagnosis of hip fracture (OR = 4.28), stroke (OR = 3.47), lower extremity arthritis (OR = 2.60), heart attack (OR = 2.29), diabetes (OR = 2.03) and obesity (OR = 1.50). Impaired lower-extremity function was significantly associated with older age (75+ years old), gender (female) and marital status (unmarried). In addition, there was a linear increase in the risk of function loss by number of medical conditions. It appears that Mexican-American elderly diagnosed with medical conditions, especially stroke and hip fracture, have a high risk for lower-extremity dysfunction. These findings have implications for efforts to prevent or reduce lower-extremity dysfunction, as well as for the provision of community-based long-term care services for Mexican-American elderly.


Development and Comparison of Dengue Vulnerability Indices Using GIS-Based Multi-Criteria Decision Analysis in Lao PDR and Thailand.
Dengue is a continuous health burden in Laos and Thailand. We assessed and mapped dengue vulnerability in selected provinces of Laos and Thailand using multi-criteria decision approaches. An ecohealth framework was used to develop dengue vulnerability indices (DVIs) that explain links between population, social and physical environments, and health to identify exposure, susceptibility, and adaptive capacity indicators. Three DVIs were constructed using two objective approaches, Shannon's Entropy (SE) and the Water-Associated Disease Index (WADI), and one subjective approach, the Best-Worst Method (BWM). Each DVI was validated by correlating the index score with dengue incidence for each spatial unit (district and subdistrict) over time. A Pearson's correlation coefficient (r) larger than 0.5 and a p-value less than 0.05 implied a good spatial and temporal performance. Spatially, DVIWADI was significantly correlated on average in 19% (4-40%) of districts in Laos (mean r = 0.5) and 27% (15-53%) of subdistricts in Thailand (mean r = 0.85). The DVISE was validated in 22% (12-40%) of districts in Laos and in 13% (3-38%) of subdistricts in Thailand. The DVIBWM was only developed for Laos because of lack of data in Thailand and was significantly associated with dengue incidence on average in 14% (0-28%) of Lao districts. The DVIWADI indicated high vulnerability in urban centers and in areas with plantations and forests. In 2019, high DVIWADI values were observed in sparsely populated areas due to elevated exposure, possibly from changes in climate and land cover, including urbanization, plantations, and dam construction. Of the three indices, DVIWADI was the most suitable vulnerability index for the study area. The DVIWADI can also be applied to other water-associated diseases, such as Zika and chikungunya, to highlight priority areas for further investigation and as a tool for prevention and interventions.


Continuous distributions of specific ventilation recovered from inert gas washout.
We describe a new technique for recovering continuous distributions of ventilation (V) as a function of tidal ventilation/volume ratio (V/V0) from the nitrogen washout. The analysis yields a continuous distribution of V as a function of V/V0 represented as fractional ventilations of 50 compartments plus dead space. The procedure was verified by recovering known distributions from data to which noise had been added. Using an apparatus to control the subject's tidal volume and FRC, mixed expired N2 data gave the following results: a) the distributions of young, normal subjects were narrow and unimodal with a mean ln standard deviation of 0.56 plus or minus 0.13; b) those of subjects over age 40 were broader (ln SD 0.86 plus or minus 0.19) with more poorly ventilated units; c) patients with pulmonary disease of all descriptions showed enlarged dead space; d) patients with cystic fibrosis showed multimodal distributions with the bulk of the ventilation going to overventilated units; and e) patients with obstructive lung diseases fell into several classes, three of which are illustrated. These results suggest that our approach is well suited for clinical investigation.


Comparative sequence, structure and redox analyses of Klebsiella pneumoniae DsbA show that anti-virulence target DsbA enzymes fall into distinct classes.
Bacterial DsbA enzymes catalyze oxidative folding of virulence factors, and have been identified as targets for antivirulence drugs. However, DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA (EcDsbA). Nonetheless, sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors, highlighting the potential to inhibit virulence across a range of organisms by targeting DsbA. For example, Salmonella enterica typhimurium (SeDsbA, 86 % sequence identity to EcDsbA) shares almost identical structural, surface and redox properties. Using comparative sequence and structure analysis we predicted that five other bacterial DsbAs would share these properties. To confirm this, we characterized Klebsiella pneumoniae DsbA (KpDsbA, 81 % identity to EcDsbA). As expected, the redox properties, structure and surface features (from crystal and NMR data) of KpDsbA were almost identical to those of EcDsbA and SeDsbA. Moreover, KpDsbA and EcDsbA bind peptides derived from their respective DsbBs with almost equal affinity, supporting the notion that compounds designed to inhibit EcDsbA will also inhibit KpDsbA. Taken together, our data show that DsbAs fall into different classes; that DsbAs within a class may be predicted by sequence analysis of binding loops; that DsbAs within a class are able to complement one another in vivo and that compounds designed to inhibit EcDsbA are likely to inhibit DsbAs within the same class.


Binding of glycosaminoglycans to sodium urate and uric acid crystals.
The binding of heparin, chondroitin sulphate and the low molecular weight heparin analogue pentosan polysulphate to sodium urate and uric acid crystals was studied by the use of radioactively labelled glycosaminoglycans were used in the binding step, and varying amounts of unlabelled glycosaminoglycans for the competition experiments. The experiments were carried out in 140 mmol/l NaCl at pH 6, with or without 5 mmol/l CaCl2 in the solution. A reversible and almost complete binding of heparin and chondroitin sulphate to sodium urate crystals did occur in the presence of calcium, whereas pentosan polysulphate bound incompletely. The binding was much less pronounced in the calcium-free conditions. Uric acid crystals did not bind any of the three inhibitors, not even with calcium present. The clinical relevance depends on whether sodium urate microcrystals are present in the urine of calcium stone patients, to cause a binding and thereby a masking or inactivation of these inhibitors in the urine, which seems to be possible in the presence of calcium. However, the potential of pentosan polysulphate for the treatment of calcium stone patients does not seem to be at risk from this effect.


Cytoskeletal Filamin A Differentially Modulates RNA Polymerase III Gene Transcription in Transformed Cell Lines.
Cytoskeletal filamin A (FLNA) is an important protein involved in multiple cellular processes. Previous studies have shown that FLNA can promote or inhibit cancer growth and development; however, the mechanisms underlying these events are not fully understood. Here we show that, in both 293T and SaOS2 cells, knockdown of FLNA significantly enhanced transcription of RNA polymerase (pol) III-transcribed genes except for a subset of tRNA genes. In contrast, re-expression of FLNA in an FLNA-deficient melanoma cell line (A7) repressed transcription of all pol III-transcribed genes, suggesting that FLNA inhibits pol III transcription in a cell type-specific manner. Chromatin immunoprecipitation assays revealed that the repression of pol III gene transcription by FLNA correlates with the decreased occupancy of the RNA pol III transcription machinery at promoters. Immunofluorescence microscopy and coimmunoprecipitation assays revealed that FLNA can associate with the RNA pol III transcription machinery through its actin-binding domain within nuclei. Mechanistic analysis revealed that FLNA suppresses pol III gene transcription by confining the recruitment of the RNA pol III transcription machinery at the promoters of the genes that are sensitive to the alteration of FLNA expression. These findings not only extend the understanding of FLNA function in cells but also provide novel insights into the mechanism by which FLNA represses cell proliferation.


Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.
The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.


Oil biosynthesis in a basal angiosperm: transcriptome analysis of Persea Americana mesocarp.
The mechanism by which plants synthesize and store high amounts of triacylglycerols (TAG) in tissues other than seeds is not well understood. The comprehension of controls for carbon partitioning and oil accumulation in nonseed tissues is essential to generate oil-rich biomass in perennial bioenergy crops. Persea americana (avocado), a basal angiosperm with unique features that are ancestral to most flowering plants, stores ~ 70 % TAG per dry weight in its mesocarp, a nonseed tissue. Transcriptome analyses of select pathways, from generation of pyruvate and leading up to TAG accumulation, in mesocarp tissues of avocado was conducted and compared with that of oil-rich monocot (oil palm) and dicot (rapeseed and castor) tissues to identify tissue- and species-specific regulation and biosynthesis of TAG in plants. RNA-Seq analyses of select lipid metabolic pathways of avocado mesocarp revealed patterns similar to that of other oil-rich species. However, only some predominant orthologs of the fatty acid biosynthetic pathway genes in this basal angiosperm were similar to those of monocots and dicots. The accumulation of TAG, rich in oleic acid, was associated with higher transcript levels for a putative stearoyl-ACP desaturase and endoplasmic reticulum (ER)-associated acyl-CoA synthetases, during fruit development. Gene expression levels for enzymes involved in terminal steps to TAG biosynthesis in the ER further indicated that both acyl-CoA-dependent and -independent mechanisms might play a role in TAG assembly, depending on the developmental stage of the fruit. Furthermore, in addition to the expression of an ortholog of WRINKLED1 (WRI1), a regulator of fatty acid biosynthesis, high transcript levels for WRI2-like and WRI3-like suggest a role for additional transcription factors in nonseed oil accumulation. Plastid pyruvate necessary for fatty acid synthesis is likely driven by the upregulation of genes involved in glycolysis and transport of its intermediates. Together, a comparative transcriptome analyses for storage oil biosynthesis in diverse plants and tissues suggested that several distinct and conserved features in this basal angiosperm species might contribute towards its rich TAG content. Our work represents a comprehensive transcriptome resource for a basal angiosperm species and provides insight into their lipid metabolism in mesocarp tissues. Furthermore, comparison of the transcriptome of oil-rich mesocarp of avocado, with oil-rich seed and nonseed tissues of monocot and dicot species, revealed lipid gene orthologs that are highly conserved during evolution. The orthologs that are distinctively expressed in oil-rich mesocarp tissues of this basal angiosperm, such as WRI2, ER-associated acyl-CoA synthetases, and lipid-droplet associated proteins were also identified. This study provides a foundation for future investigations to increase oil-content and has implications for metabolic engineering to enhance storage oil content in nonseed tissues of diverse species.


Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.
We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.


T-cell Receptors Engineered De Novo for Peptide Specificity Can Mediate Optimal T-cell Activity without Self Cross-Reactivity.
Despite progress in adoptive T-cell therapies, the identification of targets remains a challenge. Although chimeric antigen receptors recognize cell-surface antigens, T-cell receptors (TCR) have the advantage that they can target the array of intracellular proteins by binding to peptides associated with major histocompatibility complex (MHC) products (pepMHC). Although hundreds of cancer-associated peptides have been reported, it remains difficult to identify effective TCRs against each pepMHC complex. Conventional approaches require isolation of antigen-specific CD8+ T cells, followed by TCRαβ gene isolation and validation. To bypass this process, we used directed evolution to engineer TCRs with desired peptide specificity. Here, we compared the activity and cross-reactivity of two affinity-matured TCRs (T1 and RD1) with distinct origins. T1-TCR was isolated from a melanoma-reactive T-cell line specific for MART-1/HLA-A2, whereas RD1-TCR was derived de novo against MART-1/HLA-A2 by in vitro engineering. Despite their distinct origins, both TCRs exhibited similar peptide fine specificities, focused on the center of the MART-1 peptide. In CD4+ T cells, both TCRs mediated activity against MART-1 presented by HLA-A2. However, in CD8+ T cells, T1, but not RD1, demonstrated cross-reactivity with endogenous peptide/HLA-A2 complexes. Based on the fine specificity of these and other MART-1 binding TCRs, we conducted bioinformatics scans to identify structurally similar self-peptides in the human proteome. We showed that the T1-TCR cross-reacted with many of these self-peptides, whereas the RD1-TCR was rarely cross-reactive. Thus, TCRs such as RD1, generated de novo against cancer antigens, can serve as an alternative to TCRs generated from T-cell clones.


Hemispheric specialization of human inferior temporal cortex during coarse-to-fine and fine-to-coarse analysis of natural visual scenes.
Recent models of visual recognition have suggested that perceptual analysis may start with a parallel extraction of different spatial frequencies (SF), using a preferential coarse-to-fine (low-to-high SF) sequence of processing. A rapid extraction of low spatial frequency (LSF) information may thus provide an initial and crude parsing of the visual scene, subsequently refined by slow but more detailed high spatial frequency (HSF) information. However, the sequence of SF analysis could be flexible, a high-to-low (HtL) being sometimes preferred to a low-to-high (LtH) SF sequence depending on task demands. Furthermore, it has also been suggested that the right vs. left hemisphere might be differentially specialized in LSF vs. HSF analysis, respectively. By manipulating the temporal succession of LSF and HSF stimuli, the present fMRI study investigated whether such hemispheric specialization may underlie the flexible use of different time-course in SF analysis. Participants performed a matching task between two successive images of natural scenes (LSF or HSF) that were displayed either in an LtH (LSF scene presented first and HSF scene second) or in a reverse HtL sequence. A direct inter-hemispheric comparison of the neural responses evoked by each SF sequence revealed greater activations within the right occipito-temporal cortex for the LtH sequence and within the left occipito-temporal cortex for the HtL sequence. These fMRI results suggest that the hemisphere preferentially engaged during the sequential processing of different SF might be determined by the initial SF-band appearing in this sequence, and that both a coarse-to-fine and fine-to-coarse analysis might independently take place in the two hemispheres.


The 16p11.2 locus modulates brain structures common to autism, schizophrenia and obesity.
Anatomical structures and mechanisms linking genes to neuropsychiatric disorders are not deciphered. Reciprocal copy number variants at the 16p11.2 BP4-BP5 locus offer a unique opportunity to study the intermediate phenotypes in carriers at high risk for autism spectrum disorder (ASD) or schizophrenia (SZ). We investigated the variation in brain anatomy in 16p11.2 deletion and duplication carriers. Beyond gene dosage effects on global brain metrics, we show that the number of genomic copies negatively correlated to the gray matter volume and white matter tissue properties in cortico-subcortical regions implicated in reward, language and social cognition. Despite the near absence of ASD or SZ diagnoses in our 16p11.2 cohort, the pattern of brain anatomy changes in carriers spatially overlaps with the well-established structural abnormalities in ASD and SZ. Using measures of peripheral mRNA levels, we confirm our genomic copy number findings. This combined molecular, neuroimaging and clinical approach, applied to larger datasets, will help interpret the relative contributions of genes to neuropsychiatric conditions by measuring their effect on local brain anatomy.


Treatment of irritable bowel syndrome.
Irritable bowel syndrome affects 10 to 15 percent of the U.S. population to some degree. This condition is defined as abdominal pain and discomfort with altered bowel habits in the absence of any other mechanical, inflammatory, or biochemical explanation for these symptoms. Irritable bowel syndrome is more likely to affect women than men and is most common in patients 30 to 50 years of age. Symptoms are improved equally by diets supplemented with fiber or hydrolyzed guar gum, but more patients prefer hydrolyzed guar gum. Antispasmodic agents may be used as needed, but anticholinergic and other side effects limit their use in some patients. Loperamide is an option for treatment of moderately severe diarrhea. Antidepressants have been shown to relieve pain and may be effective in low doses. Trials using alosetron showed a clinically significant, although modest, gain over placebo, but it is indicated only for women with severe diarrhea-predominant symptoms or for those in whom conventional treatment has failed. Tegaserod has an advantage over placebo in constipation-predominant irritable bowel syndrome; it is indicated for up to 12 weeks of treatment in women. However, postmarketing reports of severe diarrhea and ischemic colitis further limit its use. Herbal therapies such as peppermint oil also may be effective in the treatment of irritable bowel syndrome. Therapies should focus on specific gastrointestinal dysfunctions (e.g., constipation, diarrhea, pain), and medications only should be used when nonprescription remedies do not work or when symptoms are severe.


Trend analysis and short-term forecast of incident HIV infection in Ghana.
The study uses time-series modelling to determine and predict trends in incident HIV infection in Ghana among specific age groups. The HIV data for Ghana were grouped according to northern and southern spatial sectors as they exhibited slightly different data collection formats. The trend of the epidemic is modelled using moving-average smoothing techniques, and the Box-Jenkins ARIMA model is used to forecast cases of newly acquired (incident) HIV infection. Trend analysis of past growth patterns reveals an increase in new cases of HIV infection in the northern sector, with the greatest increase occurring among persons aged 30 years and over. The epidemic in the southern sector appears to have levelled off. However, incident HIV infection in the 20-39-year-old age group of females in the sector is estimated to increase in the next three years. Moreover, the estimates suggest a higher increase in incident cases than that predicted by the National AIDS Control Programme. Nevertheless, incident HIV infection among persons aged 19 and below is found to be relatively stable. Thus, if efforts are made to reduce or prevent an increase in the number of new infections in the northern sector, and for the 20-39 years age group in the southern sector, Ghana will have a brighter future with regard to its response to the HIV epidemic. These findings can assist with developing strategic-intervention policy planning for Ghana and other countries in sub-Saharan Africa.


Specific immunoglobulin isotypes correlate with disease activity, morphology, duration and HLA association in Pemphigus vulgaris.
The molecular basis of disease heterogeneity in autoimmune conditions such as Pemphigus vulgaris is poorly understood. Although desmoglein 3 (Dsg3) has been well established as a primary target of immunoglobulin (Ig) autoantibodies in PV, there remain several questions regarding the overall distribution of anti-Dsg3 Ig subtypes among patient subsets and considerable controversy regarding whether an isotype switch can be observed between phases of disease activity. To systematically address the outstanding questions related to Ig-isotype specificity in PV, we analyzed IgA, IgM, IgG1, 2, 3 and 4 anti-Dsg3 levels by ELISA in 202 serum samples obtained from 92 patients with distinct clinical profiles based on a set of defined variable (activity, morphology, age, duration) and constant (HLA-type, gender, age of onset) clinical parameters, and 47 serum samples from HLA-matched and -unmatched controls. Our findings provide support for earlier studies identifying IgG4 and IgG1 as the predominant antibodies in PV with significantly higher levels in active than remittent patients. We do not see evidence for an isotype switch between phases of disease activity and remission, and both IgG4 and IgG1 subtypes remain elevated in remittent patients relative to controls. We do, however, find IgG4 to be the sole subtype that further distinguishes PV patient subgroups based on different disease morphologies, disease duration, and HLA-types. These data provide further insight into the immune mechanisms responsible for phenotypic expression of disease, and contribute to the broader effort to establish comprehensive immunoprofiles underlying disease heterogeneity to facilitate increasingly specific and individualized therapeutic interventions.


CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies.
Antibody-dependent cell-mediated cytotoxicity (ADCC), which is largely mediated by natural killer (NK) cells, is thought to play an important role in the efficacy of rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. CD137 is a costimulatory molecule expressed on a variety of immune cells after activation, including NK cells. In the present study, we show that an anti-CD137 agonistic mAb enhances the antilymphoma activity of rituximab by enhancing ADCC. Human NK cells up-regulate CD137 after encountering rituximab-coated tumor B cells, and subsequent stimulation of these NK cells with anti-CD137 mAb enhances rituximab-dependent cytotoxicity against the lymphoma cells. In a syngeneic murine lymphoma model and in a xenotransplanted human lymphoma model, sequential administration of anti-CD20 mAb followed by anti-CD137 mAb had potent antilymphoma activity in vivo. These results support a novel, sequential antibody approach against B-cell malignancies by targeting first the tumor and then the host immune system.


Generic task-related occupational requirements for Royal Naval personnel.
Physical tests and selection criteria have historically been used by many military organizations. However, the standards associated with them have come under increasing scrutiny in recent years. This paper describes a series of experiments that were conducted to establish task-related occupational tests and standards (TBTs) for Royal Naval (RN) personnel. A total of 172 (106 male and 66 female) RN personnel volunteered for these experiments, which were designed to: identify the anthropometric requirements for operating various safety hatches and doors on board a RN Frigate (TBT1); quantify the metabolic demands of shipboard firefighting tasks and establish an aerobic fitness standard (TBT2); and identify a battery of tests to predict performance of shipboard casualty-carrying tasks (TBT3). Whilst all subjects completed the criterion tasks during TBT1, performance of the bulkhead door (BD) escape task was related to height (r = 0.50- 0.62, P < 0.05) and vertical reach (r = 0.42-0.54, P < 0.05), with shorter subjects struggling to perform the task. During TBT2, the mean metabolic demand of representative firefighting tasks was 38 ml/min/kg, which must be sustained for 20-30 min. Finally, a battery of tests incorporating measures of lean body mass, fat mass, standing broad jump, 20 m sprint, press-ups, sit-ups and grip strength produced a high correlation (r = 0.89, P < 0.01) with casualty-carrying task performance. From the results of these experiments, it is recommended that RN personnel perform the BD simulation task at the recruitment stage (TBT1), to prove that they possess the anthropometric characteristics commensurate with survival at sea. Secondly, personnel should be frequently screened to ascertain whether they have the maximal aerobic power (41 ml/min/kg) commensurate with shipboard firefighting for 20-30 min (TBT2). Finally, they should perform the battery of proposed tests and score at least 34 points, in order to establish whether they have the anaerobic and strength capacity commensurate with shipboard casualty-carrying tasks (TBT3).


The neutral posture of the cervical spine is not unique in human subjects.
Cervical spine injuries often happen in dynamic environments (e.g., sports and motor vehicle crashes) where individuals may be moving their head and neck immediately prior to impact. This motion may reposition the cervical vertebrae in a way that is dissimilar to the upright resting posture that is often used as the initial position in cadaveric studies of catastrophic neck injury. Therefore our aim was to compare the "neutral" cervical alignment measured using fluoroscopy of 11 human subjects while resting in a neutral posture and as their neck passed through neutral during the four combinations of active flexion and extension movements in both an upright and inverted posture. Muscle activation patterns were also measured unilaterally using surface and indwelling electromyography in 8 muscles and then compared between the different conditions. Overall, the head posture, cervical spine alignment and muscle activation levels were significantly different while moving compared to resting upright. Compared to the resting upright condition, average head postures were 6-13° more extended, average vertebral angles varied from 11° more extended to 10° more flexed, and average muscle activation levels varied from unchanged to 10% MVC more active, although the exact differences varied with both direction of motion and orientation. These findings are important for ex vivo testing where the head and neck are statically positioned prior to impact - often in an upright neutral posture with negligible muscle forces - and suggest that current cadaveric head-first impact tests may not reflect many dynamic injury environments.


Wash-out kinetics and efficacy of a modified lavage technique for alveolar proteinosis.
Whole lung lavage (WLL) is the standard treatment for pulmonary alveolar proteinosis (PAP). This study aimed to provide data about the kinetics of the protein wash-out, to identify factors influencing the protein concentration in the recovered fluid and to assess the efficacy of a modified lavage technique. Samples of 180 WLLs from 42 adult PAP patients were collected. 110 WLLs were performed according to the classical technique. In 70 WLLs, repeated manual ventilation was applied during the procedure. Spectrophotometry was used to measure the protein concentration in the recovered fluid. The initial protein concentration in the recovered fluid was 460 mg · dL(-1), the final concentration was 26 mg · dL(-1) and the total amount of removed proteins during a lavage was 17.5 g. A history of dust exposure was associated with a higher residual protein concentration in the recovered fluid (p=0.000013). The amount of removed proteins correlated inversely with the diffusing capacity of the lung for carbon monoxide (p=0.001) and oxygen tension (p=0.004). The modified technique removed a greater amount of proteins than the classical technique and prolonged the time to relapse (p=0.011). Exposure to dust seems to influence the kinetics of the protein wash-out. Applying manual ventilation during the procedure can enhance the efficacy of WLL.


Coyote predation control by aversive conditioning.
Conditioned aversions were induced in coyotes by producing lithium chloride illness in them following a meal, and the effects upon eating and attack behavior were observed. One trial with a given meat and lithium is sufficient to establish a strong aversion which inhibits eating the flesh of that prey. One or two trials with a given flesh (lamb or rabbit) specifically suppresses the attack upon the averted prey but leaves the coyote free to attack the alternative prey. A method of saving both prey and predator is discussed.


Selective vulnerability of cerebellar granule neuroblasts and their progeny to drugs with abuse liability.
Cerebellar development is shaped by the interplay of genetic and numerous environmental factors. Recent evidence suggests that cerebellar maturation is acutely sensitive to substances with abuse liability including alcohol, opioids, and nicotine. Assuming substance abuse disrupts cerebellar maturation, a central question is: what are the basic mechanisms underlying potential drug-induced developmental defects? Evidence reviewed herein suggests that the maturation of granule neurons and their progeny are intrinsically affected by several classes of substances with abuse liability. Although drug abuse is also likely to target directly other cerebellar neuron and glial types, such as Purkinje cells and Bergmann glia, findings in isolated granule neurons suggest that they are often the principle target for drug actions. Developmental events that are selectively disrupted by drug abuse in granule neurons and/or their neuroblast precursors include proliferation, migration, differentiation (including neurite elaboration and synapse formation), and programmed cell death. Moreover, different classes of drugs act through distinct molecular mechanisms thereby disrupting unique aspects of development. For example, drug-induced perturbations in: (i) neurotransmitter biogenesis; (ii) ligand and ion-gated receptor function and their coupling to intracellular effectors; (iii) neurotrophic factor biogenesis and signaling; and (iv) intercellular adhesion are all likely to have significant effects in shaping developmental outcome. In addition to identifying therapeutic strategies for drug abuse intervention, understanding the mechanisms by which drugs affect cellular maturation is likely to provide a better understanding of the neurochemical events that normally shape central nervous system development.


Development of an endometriosis quality-of-life instrument: The Endometriosis Health Profile-30.
To describe a new disease-specific questionnaire with established measurement properties that addresses the dimensions of health-related quality of life considered important to women with endometriosis. The Endometriosis Health Profile-30 was developed in three stages. Stage 1 included open-ended exploratory interviews with 25 women to generate the items on the questionnaire. Stage 2 was an 87-item questionnaire administered in a postal survey to identify the most salient dimensions of health-related quality of life. In stage 3, the reliability and validity of the questionnaire were evaluated. The final instrument contained a core questionnaire with 30 items and five scales: pain, control and powerlessness, emotional well-being, social support, and self-image. Six modular parts consisting of 23 questions were also developed and measured the areas of sexual intercourse, work, relationship with children, feelings about the medical profession, treatment, and infertility. All the scales achieved high internal reliability, with Cronbach's alpha coefficient ranging from 0.83 to 0.93 (core questionnaire) and 0.79 to 0.96 (modules). The intraclass correlation coefficients to evaluate the test-retest reliability were high (range 0.88-0.98, P <.001). Content validity was demonstrated as the questionnaire was developed from interviews of patients rather than existing literature and clinical scales. Construct validity was assessed by correlating the Endometriosis Health Profile-30 scales with the relevant Short Form-36 scales. High correlations for all comparisons were found (-0.41 to -0.73). The Endometriosis Health Profile-30 is a reliable, valid, patient-generated instrument to measure the health-related quality of life of women with endometriosis. Its application in various health care settings will provide new and valuable information on the effect of endometriosis on health-related quality of life from the patients' perspective.


Built and socioeconomic environments: patterning and associations with physical activity in U.S. adolescents.
Inter-relationships among built and socioeconomic environmental characteristics may result in confounding of associations between environment exposure measures and health behaviors or outcomes, but traditional multivariate adjustment can be inappropriate due to collinearity. We used principal factor analysis to describe inter-relationships between a large set of Geographic Information System-derived built and socioeconomic environment measures for adolescents in the National Longitudinal Study of Adolescent Health (Wave I, 1995-96, n = 17,294). Using resulting factors in sex-stratified multivariate negative binomial regression models, we tested for confounding of associations between built and socioeconomic environment characteristics and moderate to vigorous physical activity (MVPA). Finally, we used knowledge gained from factor analysis to construct replicable environmental measures that account for inter-relationships and avoid collinearity. Using factor analysis, we identified three built environment constructs [(1) homogenous landscape; 2) development intensity with high pay facility count; 3) development intensity with high public facility count] and two socioeconomic environment constructs [1) advantageous economic environment, 2) disadvantageous social environment]. In regression analysis, confounding of built environment-MVPA associations by socioeconomic environment factors was stronger than among built environment factors. In fully adjusted models, MVPA was negatively associated with the highest (versus lowest) quartile of homogenous land cover in males [exp(coeff) (95% CI): 0.91 (0.86, 0.96)] and intensity (pay facilities) [exp(coeff) (95% CI): 0.92 (0.85, 0.99)] in females. Single proxy measures (Simpson's diversity index, count of pay facilities, count of public facilities, median household income, and crime rate) representing each environmental construct replicated associations with MVPA. Environmental characteristics are inter-related. Both built and SES environments should be incorporated into analysis in order to minimize confounding. Single environmental measures may be useful proxies for environmental constructs in longitudinal analysis and replication in external populations, but more research is needed to better understand mechanisms of action, and ultimately identify policy-relevant environmental determinants of physical activity.


Undiagnosed Obstructive Sleep Apnea and Physical Activity in Older Manual Workers.
Cardiovascular disease is a negative health outcome of obstructive sleep apnea (OSA). Risk factors associated with OSA development include low physical activity (PA), high body mass index (BMI), and increasing age (>50 years), and weight loss is usually recommended as treatment. This cross-sectional study examined the association between PA, BMI, and OSA severity in manual workers. Fifty-five participants (23 females and 32 males; mean age 55.2 years), were examined for OSA and completed a PA and anthropometric assessment. On average, OSA severity was mild, PA levels were moderate, and 32% of the sample was classified as obese. PA was negatively associated with OSA severity, but BMI strongly independently predicted OSA severity, with no evidence of mediation. As both PA and BMI were significantly associated with OSA in older manual workers, increasing PA should also be a focus of treatment for OSA.


Hemangiopericytoma invading the craniovertebral junction: First reported case and review of the literature.
Occurrence of hemangiopericytoma (HPC) in the central nervous system is rare. Spinal HPCs with intramedullary involvement are even more unusual. We present a case of a craniovertebral intradural HPC with both extra- and intra-medullary extensions. Though the patient presented with vague cervical symptoms, imaging was indicative of an intradural lesion from the occiput to C4 and a second smaller, subclinical lesion, at the T2-3 level. He underwent gross total surgical resection of the craniovertebral lesion and did well post-operatively. The thoracic lesion was treated with radiosurgery and the patient is neurologically at baseline 5 years later. Gross total resection of HPCs is the recommended treatment when possible. Histopathology is crucial for diagnosis due to both its rarity and similar characteristics to other tumors on physical and radiographic evaluations. Recognizing that these uncommon tumors can occur with both extra-medullary and intra-medullarly locations are important for diagnosis and treatment recommendations. Future studies using national surgical databases that contain histology will be needed to understand the long-term clinical outcomes.


FTY720-enhanced T cell homing is dependent on CCR2, CCR5, CCR7, and CXCR4: evidence for distinct chemokine compartments.
FTY720 stimulates CCR7-driven T cell homing to peripheral lymph nodes (LN) by direct activation of sphingosine 1-phosphate receptors, along with the participation of multidrug transporters, 5-lipoxygenase, and G protein-coupled receptors for chemokines. In this study, we demonstrate that FTY720 also directly stimulates in vitro T cell chemotaxis to CCR2-CCL2, but not to a variety of other chemokines, including CCR5-CCL3/4/5 and CXCR4-CXCL12. FTY720 influences CCR2-CCL2-driven migration through activation of the multidrug transporters, Abcb1 and Abcc1, and through 5-lipoxygenase activity. In vivo administration of FTY720 induces chemokine-dependent migration of T cells in the thymus, peripheral blood, LN, and spleen. The CCR7 and CCR2 chemokine ligands are required for both T cell sequestration in LN and thymic T cell egress following FTY720 administration. Furthermore, FTY720 administration uncovers a requirement for CXCR4 ligands for LN homing, but not for thymic egress, and CCR5 for thymic egress, but not LN homing. FTY720-driven splenic and peripheral blood T cell egress are both independent of CCR2, CCR5, CCR7, or CXCR4. These results indicate that FTY720- and sphingosine 1-phosphate receptor-stimulated T cell migration are dependent on the restricted usage of chemokine receptor-ligand pairs within discrete anatomic compartments.


Parametric modeling of quantile regression coefficient functions.
Estimating the conditional quantiles of outcome variables of interest is frequent in many research areas, and quantile regression is foremost among the utilized methods. The coefficients of a quantile regression model depend on the order of the quantile being estimated. For example, the coefficients for the median are generally different from those of the 10th centile. In this article, we describe an approach to modeling the regression coefficients as parametric functions of the order of the quantile. This approach may have advantages in terms of parsimony, efficiency, and may expand the potential of statistical modeling. Goodness-of-fit measures and testing procedures are discussed, and the results of a simulation study are presented. We apply the method to analyze the data that motivated this work. The described method is implemented in the qrcm R package.


Inhibition of radiation-induced G2 delay potentiates cell death by apoptosis and/or the induction of giant cells in colorectal tumor cells with disrupted p53 function.
We have previously identified a p53-independent apoptotic response that is delayed until 48-72 h after irradiation of colorectal adenoma and carcinoma cells. Because the delay appears to be in part due to a transient G2 cell cycle arrest, the importance of this checkpoint in the mechanism of ionizing radiation (IR)-induced death of colorectal tumor cells was investigated. An adenoma cell line with (282Arg-->Trp) mutant p53 (S/RG/C2) and a carcinoma cell line (PC/JW/FI) lacking p53 protein treated with 5 Gy IR in the presence of 1.5 mm caffeine (CAF) reduced IR-induced G2 arrest and increased the level of apoptosis (1.5-1.6-fold) 24 h after treatment. Increased IR apoptotic cell death with CAF significantly reduced IR cell survival over a 7-day period in S/RG/C2 and PC/JW/FI. To investigate whether CAF radiosensitization correlated with lack of wild-type (wt) p53, we studied transfected derivatives of an adenoma-derived cell line (PC/AA/C1), in which the endogenous wt p53 activity was disrupted by the expression of a dominant negative (273Arg-->His) p53 mutant protein (designated AA/273p53/B). This p53-defective cell line was also radiosensitized by CAF, whereas the vector control (AA/PCMV/D), which retained wt p53 activity, was not. In addition, as with the S/RG/C2 and PC/JW/FI cell lines, the 7-day IR cell survival was reduced significantly in AA/273p53/B compared with the vector control cell line. This suggests that radiosensitization by CAF and increased cell death is dependent on loss of wt p53 function. Interestingly, radiosensitization of the AA/273p53/B cell line was not associated with accelerated apoptosis but correlated with increased polyploid giant cells, which have been associated with disruption of cell cycle checkpoints and genomic instability. These results demonstrate that G2 checkpoint inhibition with CAF leads to preferential IR cell killing in cell lines in which wt p53 is inactivated and that this increased cell killing is not necessarily dependent on increased IR-induced apoptosis.


Lobe specific Ca2+-calmodulin nano-domain in neuronal spines: a single molecule level analysis.
Calmodulin (CaM) is a ubiquitous Ca(2+) buffer and second messenger that affects cellular function as diverse as cardiac excitability, synaptic plasticity, and gene transcription. In CA1 pyramidal neurons, CaM regulates two opposing Ca(2+)-dependent processes that underlie memory formation: long-term potentiation (LTP) and long-term depression (LTD). Induction of LTP and LTD require activation of Ca(2+)-CaM-dependent enzymes: Ca(2+)/CaM-dependent kinase II (CaMKII) and calcineurin, respectively. Yet, it remains unclear as to how Ca(2+) and CaM produce these two opposing effects, LTP and LTD. CaM binds 4 Ca(2+) ions: two in its N-terminal lobe and two in its C-terminal lobe. Experimental studies have shown that the N- and C-terminal lobes of CaM have different binding kinetics toward Ca(2+) and its downstream targets. This may suggest that each lobe of CaM differentially responds to Ca(2+) signal patterns. Here, we use a novel event-driven particle-based Monte Carlo simulation and statistical point pattern analysis to explore the spatial and temporal dynamics of lobe-specific Ca(2+)-CaM interaction at the single molecule level. We show that the N-lobe of CaM, but not the C-lobe, exhibits a nano-scale domain of activation that is highly sensitive to the location of Ca(2+) channels, and to the microscopic injection rate of Ca(2+) ions. We also demonstrate that Ca(2+) saturation takes place via two different pathways depending on the Ca(2+) injection rate, one dominated by the N-terminal lobe, and the other one by the C-terminal lobe. Taken together, these results suggest that the two lobes of CaM function as distinct Ca(2+) sensors that can differentially transduce Ca(2+) influx to downstream targets. We discuss a possible role of the N-terminal lobe-specific Ca(2+)-CaM nano-domain in CaMKII activation required for the induction of synaptic plasticity.


Neuromuscular anatomy and evolution of the cetacean forelimb.
The forelimb of cetaceans (whales, dolphins, and porpoises) has been radically modified during the limb-to-flipper transition. Extant cetaceans have a soft tissue flipper encasing the manus and acting as a hydrofoil to generate lift. The neuromuscular anatomy that controls flipper movement, however, is poorly understood. This study documents flipper neuromuscular anatomy and tests the hypothesis that antebrachial muscle robustness is related to body size. Data were gathered during dissections of 22 flippers, representing 15 species (7 odontocetes, 15 mysticetes). Results were compared with published descriptions of both artiodactyls and secondarily aquatic vertebrates. Results indicate muscle robustness is best predicted by taxonomic distribution and is not a function of body size. All cetaceans have atrophied triceps muscles, an immobile cubital joint, and lack most connective tissue structures and manus muscles. Forelimbs retain only three muscle groups: triceps (only the scapular head is functional as the humeral heads are vestigal), and antebrachial extensors and flexors. Well-developed flexor and extensor muscles were found in mysticetes and basal odontocetes (i.e., physeterids, kogiids, and ziphiids), whereas later diverging odontocetes (i.e., monodontids, phocoenids, and delphinids) lack or reduce these muscles. Balaenopterid mysticetes (e.g., fin and minke whales) may actively change flipper curvature, while basal odontocetes (e.g., sperm and beaked whales) probably stiffen the flipper through isometric contraction. Later diverging odontocetes lack musculature supporting digital movements and are unable to manipulate flipper curvature. Cetacean forelimbs are unique in that they have lost agility and several soft tissue structures, but retain sensory innervations.


Decelerating growth in tropical forest trees.
The impacts of global change on tropical forests remain poorly understood. We examined changes in tree growth rates over the past two decades for all species occurring in large (50-ha) forest dynamics plots in Panama and Malaysia. Stem growth rates declined significantly at both forests regardless of initial size or organizational level (species, community or stand). Decreasing growth rates were widespread, occurring in 24-71% of species at Barro Colorado Island, Panama (BCI) and in 58-95% of species at Pasoh, Malaysia (depending on the sizes of stems included). Changes in growth were not consistently associated with initial growth rate, adult stature, or wood density. Changes in growth were significantly associated with regional climate changes: at both sites growth was negatively correlated with annual mean daily minimum temperatures, and at BCI growth was positively correlated with annual precipitation and number of rainfree days (a measure of relative insolation). While the underlying cause(s) of decelerating growth is still unresolved, these patterns strongly contradict the hypothesized pantropical increase in tree growth rates caused by carbon fertilization. Decelerating tree growth will have important economic and environmental implications.


Tofupill/Femarelle (DT56a): a new phyto-selective estrogen receptor modulator-like substance for the treatment of postmenopausal bone loss.
To evaluate the efficacy of Tofupill/Femarelle (DT56a), a novel phyto-selective estrogen receptor modulator (SERM), in preserving bone mineral density (BMD) in postmenopausal women. The study sample consisted of 98 healthy, postmenopausal women who were randomly allocated, on a double-blind basis, to receive either 644 mg/d DT56a (study group) or 344 mg/d DT56a supplemented with calcium (low-dose group) for 12 months. Each participant was assessed with a comprehensive health questionnaire, a detailed physical, and laboratory and pelvic sonogram examinations at entry and every 3 months thereafter. BMD was assessed by dual-energy x-ray absorptiometry (Lunar) of the lumbar spine and femoral neck before the study began and after 12 months of treatment. After 12 months of treatment, BMD had increased in the study group by 3.6% in the lumbar spine (P = 0.039) and by 2.0% in the femoral neck (NS). In the low-dose group, BMD had decreased in the lumbar spine by 0.6% (NS) and by 0.6% in the femoral neck (NS). Comparison of the change in bone density between the groups yielded a significant difference for the lumbar spine (P = 0.037). Neither group showed a change in endometrial thickness and sex hormone levels nor reported any side effects of treatment. Tofupill treatment in postmenopausal women increases BMD without unwanted estrogenic effect. Tofupill appears to be a promising phyto-SERM for the prevention of postmenopausal osteoporosis.


Prolyl 3-hydroxylase 1, enzyme characterization and identification of a novel family of enzymes.
The collagen prolyl hydroxylases are enzymes that are required for proper collagen biosynthesis, folding, and assembly. They reside within the endoplasmic reticulum and belong to the group of 2-oxoglutarate and iron-dependent dioxygenases. Although prolyl 4-hydroxylase has been characterized as an alpha2beta2 tetramer in which protein disulfide isomerase is the beta subunit with two different alpha subunit isoforms, little is known about the enzyme prolyl 3-hydroxylase (P3H). It was initially characterized and shown to have an enzymatic activity distinct from that of prolyl 4-hydroxylase, but no amino acid sequences or genes were ever reported for the mammalian enzyme. Here we report the characterization of a novel prolyl 3-hydroxylase enzyme isolated from embryonic chicks. The primary structure of the enzyme, which we now call P3H1, demonstrates that P3H1 is a member of a family of prolyl 3-hydroxylases, which share the conserved residues present in the active site of prolyl 4-hydroxylase and lysyl hydroxylase. P3H1 is the chick homologue of mammalian leprecan or growth suppressor 1. Two other P3H family members are the genes previously called MLAT4 and GRCB. In this study we demonstrate prolyl 3-hydroxylase activity of the purified enzyme P3H1 on a full-length procollagen substrate. We also show it to specifically interact with denatured collagen and to exist in a tight complex with other endoplasmic reticulum-resident proteins. Immunohistochemistry with a monoclonal antibody specific for chick P3H1 localizes P3H1 specifically to tissues that express fibrillar collagens, suggesting that other P3H family members may be responsible for modifying basement membrane collagens.


Risk Assessment of Miners' Unsafe Behaviors: A Case Study of Gas Explosion Accidents in Coal Mine, China.
China's coal mine production situation is grim and various types of accidents occur frequently, and gas explosion accidents are the highest incidence of coal mine accidents. The authors selected 200 gas explosion accidents of coal mine enterprises in recent years, and extracted a large number of workers' unsafe behaviors. Meanwhile, four working types related to gas explosion accidents were obtained, namely ventilation, gas prevention and fire extinguishing, blasting, and electrician. This article listed some influencing factors of unsafe behaviors and corrected the probability of unsafe behaviors. In addition, a probabilistic risk assessment model was established, and the Monte Carlo method was used to analyze the risks caused by unsafe behaviors of various working types. The results show that the risk of unsafe behaviors caused by the ventilation working type is the highest, followed by gas prevention and fire extinguishing, and finally blasting and electrician. This paper studies the influencing factors of miners' unsafe behaviors from the perspective of behavior, guarantees effectively the safety management of coal mine enterprises, and lays a foundation for studying unsafe behaviors related to coal mine gas explosions.