Intact Site Research Articles

Clinical Significance of TDP-43 Neuropathology in Amyotrophic Lateral Sclerosis.

To determine the significance of TAR DNA binding protein 43 kDa (TDP-43) pathology in amyotrophic lateral sclerosis (ALS), we examined the whole brains and spinal cords of 57 patients (35 men; 22 women; mean age 63.3 years; 15 patients with c9orf72-associated ALS [c9ALS]). TDP-43 pathologic burden was determined relative to symptom onset site, disease duration, progression rate, cognitive status, and c9ALS status. There was a trend for greater TDP-43 pathologic burden in cognitively impaired patients (p = 0.07), though no association with disease duration or progression rate was seen. Shorter disease duration (p = 0.0016), more severe striatal pathology (p = 0.0029), and a trend toward greater whole brain TDP-43 pathology (p = 0.059) were found in c9ALS. Cluster analysis identified “TDP43-limited,” “TDP43-moderate,” and “TDP43-severe” subgroups. The TDP43-limited group contained more cognitively intact (p = 0.005) and lower extremity onset site (p = 0.019) patients, while other subgroups contained more cognitively impaired patients. We conclude that TDP-43 pathologic burden in ALS is associated with cognitive impairment and c9ALS, but not duration of disease or rate of progression. Further, we demonstrate a subgroup of patients with low TDP-43 burden, lower extremity onset, and intact cognition, which requires further investigation.

Idaho beginnings: A review of the evidence

Evidence sought here for a pre-Clovis presence in the Snake River basin of Idaho and northeastern Oregon in the millennium ending at 11,600 yr BP includes radiocarbon and tephrochronological dates, extinct Pleistocene faunal associations, depositional context and living floor and feature integrity, and typologically or technologically legible lithic industries. Results of a review of eleven sites found ambiguous evidence for pre-Clovis hearths in two caves, but no convincing support for the temporal priority of the Western Stemmed over the Clovis tradition. However, intact sites of the Western Stemmed tradition, dating to 10,200–9800 yr BP, survive at 4 + m below surface in deep Quaternary alluvium in canyon floors in west-central Idaho below 610 m elevation. Deeper and earlier deposits may be found in the same settings.

An Information Technology Framework for the Development of an Embedded Computer System for the Remote and Non-Destructive Study of Sensitive Archaeology Sites

The paper proposes an information technology framework for the development of an embedded remote system for non-destructive observation and study of sensitive archaeological sites. The overall concept and motivation are described. The general hardware layout and software configuration are presented. The paper concentrates on the implementation of the following informational technology components: (a) a geographically unique identification scheme supporting a global key space for a key-value store; (b) a common method for octree modeling for spatial geometrical models of the archaeological artifacts, and abstract object representation in the global key space; (c) a broadcast of the archaeological information as an Extensible Markup Language (XML) stream over the Web for worldwide availability; and (d) a set of testing methods increasing the fault tolerance of the system. This framework can serve as a foundation for the development of a complete system for remote archaeological exploration of enclosed archaeological sites like buried churches, tombs, and caves. An archaeological site is opened once upon discovery, the embedded computer system is installed inside upon a robotic platform, equipped with sensors, cameras, and actuators, and the intact site is sealed again. Archaeological research is conducted on a multimedia data stream which is sent remotely from the system and conforms to necessary standards for digital archaeology.

Upregulation of maspin expression in human cervical carcinoma cells by transforming growth factor β1 through the convergence of Smad and non-Smad signaling pathways.

Mammary serine protease inhibitor (maspin), encoded by the serpin family B member 5 gene, serves as a tumor suppressor through the inhibition of cancer cell invasion and metastasis. Paradoxically, maspin levels are upregulated in a number of types of malignant cells. Therefore, the regulation of maspin expression may depend on the genetic or epigenetic background and the specific microenvironment of carcinoma cells. In the present study, it was demonstrated that transforming growth factor β1 (TGF-β1) induced maspin expression at the transcript and protein levels in the human cervical carcinoma HeLa and human oral squamous carcinoma HSC4 cell lines. The inhibition of the mothers against decapentaplegic homolog (Smad)-dependent pathway by a Smad3-specific inhibitor suppressed maspin induction by TGF-β1 in HeLa cells. Inhibition of the non-Smad pathway by pretreatment with the mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126, or the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB202190, attenuated the effect of TGF-β1 on maspin upregulation, whereas pretreatment with pyrrolidine dithiocarbamate (a nuclear factor κB inhibitor), wortmannin (a phosphoinositide 3-kinase inhibitor) or SP600125 (a c-Jun N-terminal kinase inhibitor) did not. Notably, none of these inhibitors eliminated the TGF-β1-induced phosphorylation of Smad2. In addition, mutations at p53-binding sites in the maspin promoter suppressed TGF-β1-induced maspin expression, indicating the necessity of intact p53-binding sites on the maspin promoter. In summary, the induction of maspin expression in HeLa cells requires the convergence of TGF-β1-induced Smad and non-Smad signaling pathways, in which the latter acts via the intermediate signaling molecules MEK1/2 and p38 MAPK.

ZNF395 Is an Activator of a Subset of IFN-Stimulated Genes.

Activation of the interferon (IFN) pathway in response to infection with pathogens results in the induction of IFN-stimulated genes (ISGs) including proinflammatory cytokines, which mount the proper antiviral immune response. However, aberrant expression of these genes is pathogenic to the host. In addition to IFN-induced transcription factors non-IFN-regulated factors contribute to the transcriptional control of ISGs. Here, we show by genome wide expression analysis, siRNA-mediated suppression and Doxycycline-induced overexpression that the cellular transcription factor ZNF395 activates a subset of ISGs including the chemokines CXCL10 and CXCL11 in keratinocytes. We found that ZNF395 acts independently of IFN but enhances the IFN-induced expression of CXCL10 and CXCL11. Luciferase reporter assays revealed a requirement of intact NFκB-binding sites for ZNF395 to stimulate the CXCL10 promoter. The transcriptional activation of CXCL10 and CXCL11 by ZNF395 was abolished after inhibition of IKK by BMS-345541, which increased the stability of ZNF395. ZNF395 encodes at least two motifs that mediate the enhanced degradation of ZNF395 in response to IKK activation. Thus, IKK is required for ZNF395-mediated activation of transcription and enhances its turn-over to keep the activity of ZNF395 low. Our results support a previously unrecognized role of ZNF395 in the innate immune response and inflammation.

Eight on 80-Ruptured and Intact Multiple Site Aneurysms: Aorto-Bi-Common and External Iliac, Right Popliteal and Left Femoral and Popliteal Artery Aneurysms, Sequentially Repaired Successfully in an Octogenarian

Degenerative aortic and peripheral aneurysms are well known to present in the elderly at multiple sites at varying time. Aneurysms occurring in the arteries of the lower limb are most common after aneurysms of the infrarenal aorta. A strong association exists between the presence of true aneurysms of the femoral or popliteal arteries and those of the contralateral extremity and more importantly aortoiliac domain. Consequently, the discovery of a lower extremity aneurysm mandates careful observation, evaluation, or both for associated aneurysms in the opposite limb and abdominal aorta. Traditional open surgery is used to be mandated in ruptured aneurysm in previous years, whereas endovascular therapeutic modality is a mainstay in the current practice, especially in elderly and frail patients with short life expectancy. Herein, we present a report of successful outcome following open and endovascular repair of eight aneurysms in an octogenarian at varying times under a decade.

CREM Alpha Enhances IL-21 Production in T Cells In Vivo and In Vitro.

The cAMP-responsive element modulator alpha (CREMα) plays a role in autoimmunity and, in particular, in systemic lupus erythematosus. CREMα negatively regulates IL-2 transcription and activates IL-17 expression by direct transcriptional mechanisms. To understand the role of CREM in autoimmunity, we recently generated a mouse with a transgenic overexpression of CREMα selectively in T cells. This mouse is characterized by enhanced IL-17 and IL-21 expression. We, herein, dissect the transcriptional mechanisms of enhanced IL-21 transcription in these mice. T cells of CREMα transgenic mice display an enhanced binding of CREMα to the CD3ζ chain promoter resulting in decreased CD3ζ chain expression. This is accompanied by a decreased excitation threshold and enhanced Ca2+ influx, which is known to induce IL-21 expression via NFATc2 activation. However, CREMα directly binds to cAMP-response element (CRE) half-site within the Il-21 promoter, which results in enhanced promoter activity shown by promoter reporter assays. CREMα-induced IL-21 transcription is not abrogated in the presence of cyclosporine A but depends on an intact CRE site within the IL-21 promoter, which suggests that CREM largely enhances IL-21 expression by direct transcriptional regulation. IL-21 transcription is critical for IL-17 generation in these mice, since IL-21 receptor blockade downregulates IL-17 transcription to wild-type levels. Finally, this is of functional relevance since CREMα transgenic mice display enhanced disease activity in dextran sodium sulfate-induced colitis accompanied by higher local IL-21 expression. Thus, we describe two novel mechanisms of CREMα-dependent IL-21 transcription. Since T cells of systemic lupus erythematosus patients are characterized by enhanced IL-21 transcription, this might also be of functional relevance in humans.

Fto Plays an Oncogenic Role in Acute Myeloid Leukemia As a N6-Methyladenosine RNA Demethylase

IntroductionN6-methyladenosine (m6A) is the most abundant internal modification in messenger RNA (mRNA) mainly occurring at consensus motif of G[G>A]m6AC[U>A>C]. Despite the functional importance of m6A modification in various fundamental bioprocesses, the studies of m6A modification in cancer, especially in leukemia have largely been limited.Fat mass and obesity-associated protein (FTO), the first RNA demethylase,was known to be robustly associated with increased body mass and obesity in humans. However, the impact of FTO, especially as a RNA demethylase, in cancer development and progression has yet to be investigated. Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with distinct geneticabnormalities and variable response to treatment.Here, we aim to definethe roleof FTO as an m6A demethylase in AML.MethodsTo access the potential effect of FTO, we analyzed its expression in AML patients with distinct genetic mutations. To determine the influence of FTO on transformation capacity/ cell viability and leukemogenesis, colony-forming/replating assay (CFA), MTT assays, cell apoptosis and bone marrow transplantation (BMT) were carried out. To identify potential targets of FTO, transcriptome-wide m6A-seq and RNA-seq were performed. To evaluate the function of FTO on m6A modification and mRNA metabolism,m6A dot blot, gene-specific m6A qPCR assays and RNA stability assays were conducted. To elucidate whether FTO-mediated regulation of its targets depends on its demethylase activity, gene-specific m6A qPCR assays and luciferase reporter and mutagenesis assays were carried out. To investigate the potential roles of FTO and its targets in hematopoiesis, ATRA-induced APL cell differentiation was used.ResultsIn analysis of AML datasets, we found FTO is highly expressed in AMLs with t(11q23)/MLL-rearrangements, t(15;17)/PML-RARA, FLT3-ITD and/or NPM1 mutations. Lentivirus-induced expression of wild-type FTO, but not mutant FTO (carrying two point mutations, H231A and D233A , which disrupt its enzymatic activity), significantly enhanced colony forming activities, promoted cell proliferation/transformation, restricted cell apoptosis and decreased global mRNA m6A levelin vitro. Forced expression of Fto significantly (p<0.05; log-rank test) accelerated MLL-AF9-induced leukemogenesis and decreased global m6A level in leukemic BM cells. The opposite is true when FTO/Fto was knocked down by shRNAs or genetically knocked out.Via transcriptome-wide m6A-sequencingand RNA-sequencing (RNA-Seq) assays in MONOMAC-6 AML cells with or without overexpression or knockdown of FTO, we identified two functionally critical targets of FTO, ASB2 and RARA.Forced expression of wild-type FTO, but not mutant FTO, reduced expression of RARA and ASB2. Forced expression of either ASB2 or RARA largely recapitulated the phenotypes caused by FTO knockdown. Moreover, the effects of overexpression or knockdownof FTO can be largely rescued by that of RARA or ASB2, indicating that they are functional important targets of FTO.Forced expression and knockdown of FTO reduced and increased, respectively, the m6A levels on ASB2 and RARA mRNA transcripts,and shortened and prolonged, respectively, the half-life of ASB2 and RARAmRNA transcripts in AML cells.Importantly, FTO reduced luciferase activity ofASB23'UTR, RARA3'UTR or RARA5'UTR constructs with intact m6A sites, while mutations in the m6A sites abrogated the inhibition, demonstrating that FTO-mediated gene regulation relies on its demethylase activity.Upon ATRA treatment, FTO was significantly down-regulated, while RARA and ASB2were up-regulated in NB4 APL cells. Forced expression of FTO noticeably suppressed, while depletion of FTO enhanced, ATRA-induced cell differentiation.Forced expression of either RARA or ASB2 could also substantially enhance NB4 cell differentiation.ConclusionsIn summary, we provide compelling in vitro and in vivo evidence demonstrating that FTO, an m6A demethylase, plays a critical oncogenic role in cell transformation and leukemogenesis as well as in ATRA-mediated differentiation of leukemic cells, through reducing m6A levels in mRNA transcripts of its critical target genes such as ASB2 and RARA and thereby triggering corresponding signaling cascades. Our study highlights the functional importance of the m6A modification machinery in leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Fine and Coarse-Scale Patterns of Vegetation Diversity on Reclaimed Surface Mine-land Over a 40-Year Chronosequence.

Rangelands are described as heterogeneous, due to patterning in species assemblages and productivity that arise from species dispersal and interactions with environmental gradients and disturbances across multiple scales. The objectives of rangeland reclamation are typically vegetation establishment, plant community productivity, and soil stability. However, while fine-scale diversity is often promoted through species-rich seed mixes, landscape heterogeneity and coarse-scale diversity are largely overlooked. Our objectives were to evaluate fine and coarse-scale vegetation patterns across a 40-year reclamation chronosequence on reclaimed surface coalmine lands. We hypothesized that both α-diversity and β-diversity would increase and community patch size and species dissimilarity to reference sites would decrease on independent sites over 40 years. Plant communities were surveyed on 19 post-coalmine reclaimed sites and four intact native reference sites in central North Dakota mixed-grass prairie. Our results showed no differences in α or β-diversity and plant community patch size over the 40-year chronosequence. However, both α-diversity and β-diversity on reclaimed sites was similar to reference sites. Native species establishment was limited due to the presence of non-native species such as Kentucky bluegrass (Poa pratensis) on both the reclaimed and reference sites. Species composition was different between reclaimed and reference sites and community dissimilarity increased on reclaimed sites over the 40-year chronosequence. Plant communities resulting from reclamation followed non-equilibrium succession, even with consistent seeds mixes established across all reclaimed years. This suggests post-reclamation management strategies influence species composition outcomes and land management strategies applied uniformly may not increase landscape-level diversity.

Safety and Wound Outcomes Following Genetically Corrected Autologous Epidermal Grafts in Patients With Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating, often fatal, inherited blistering disorder caused by mutations in the COL7A1 gene encoding type VII collagen. Support and palliation are the only current therapies. To evaluate the safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with RDEB. Single-center phase 1 clinical trial conducted in the United States of 4 patients with severe RDEB with a measured area of wounds suitable for grafting of at least 100 cm2. Patients with undetectable type VII collagen keratinocyte expression were excluded. Autologous keratinocytes isolated from biopsy samples collected from 4 patients with RDEB were transduced with good manufacturing practice-grade retrovirus carrying full-length human COL7A1 and assembled into epidermal sheet grafts. Type VII collagen gene-corrected grafts (approximately 35 cm2) were transplanted onto 6 wounds in each of the patients (n = 24 grafts). The primary safety outcomes were recombination competent retrovirus, cancer, and autoimmune reaction. Molecular correction was assessed as type VII collagen expression measured by immunofluorescence and immunoelectron microscopy. Wound healing was assessed using serial photographs taken at 3, 6, and 12 months after grafting. The 4 patients (mean age, 23 years [range, 18-32 years]) were all male with an estimated body surface area affected with RDEB of 4% to 30%. All 24 grafts were well tolerated without serious adverse events. Type VII collagen expression at the dermal-epidermal junction was demonstrated on the graft sites by immunofluorescence microscopy in 9 of 10 biopsy samples (90%) at 3 months, in 8 of 12 samples (66%) at 6 months, and in 5 of 12 samples (42%) at 12 months, including correct type VII collagen localization to anchoring fibrils. Wounds with recombinant type VII collagen graft sites displayed 75% or greater healing at 3 months (21 intact graft sites of 24 wound sites; 87%), 6 months (16/24; 67%), and 12 months (12/24; 50%) compared with baseline wound sites. In this preliminary study of 4 patients with RDEB, there was wound healing in some type VII collagen gene-corrected grafts, but the response was variable among patients and among grafted sites and generally declined over 1 year. Long-term follow-up is necessary for these patients, and controlled trials are needed with a broader range of patients to better understand the potential long-term efficacy of genetically corrected autologous epidermal grafts. clinicaltrials.gov Identifier: NCT01263379.

Transition metal ion FRET uncovers K+ regulation of a neurotransmitter/sodium symporter.

Neurotransmitter/sodium symporters (NSSs) are responsible for Na+-dependent reuptake of neurotransmitters and represent key targets for antidepressants and psychostimulants. LeuT, a prokaryotic NSS protein, constitutes a primary structural model for these transporters. Here we show that K+ inhibits Na+-dependent binding of substrate to LeuT, promotes an outward-closed/inward-facing conformation of the transporter and increases uptake. To assess K+-induced conformational dynamics we measured fluorescence resonance energy transfer (FRET) between fluorescein site-specifically attached to inserted cysteines and Ni2+ bound to engineered di-histidine motifs (transition metal ion FRET). The measurements supported K+-induced closure of the transporter to the outside, which was counteracted by Na+ and substrate. Promoting an outward-open conformation of LeuT by mutation abolished the K+-effect. The K+-effect depended on an intact Na1 site and mutating the Na2 site potentiated K+ binding by facilitating transition to the inward-facing state. The data reveal an unrecognized ability of K+ to regulate the LeuT transport cycle.

Differences in vector-genome processing and illegitimate integration of non-integrating lentiviral vectors.

A variety of mutations in lentiviral vector expression systems have been shown to generate a non-integrating phenotype. We studied a novel 12 base-pair U3-LTR integrase attachment site deletion (U3-LTR att site) mutant and found similar physical titers to the previously reported integrase catalytic core mutant IN/D116N. Both mutations led to a greater than two log reduction in vector integration; with IN/D116N providing lower illegitimate integration frequency, while the U3-LTR att site mutant provided a higher level of transgene expression. The improved expression of the U3-LTR att site mutant could not be explained solely based on an observed modest increase in integration frequency. In evaluating processing, we noted significant differences in unintegrated vector forms, with the U3-LTR att site mutant leading to a predominance of 1-LTR circles. The mutations also differed in the manner of illegitimate integration. The U3-LTR att site mutant vector demonstrated integrase-mediated integration at the intact U5-LTR att site and non-integrase mediated integration at the mutated U3-LTR att site. Finally, we combined a variety of mutations and modifications and assessed transgene expression and integration frequency to show that combining modifications can improve the potential clinical utility of non-integrating lentiviral vectors.

Fetal Myelomeningocele Repair through a Mini-Hysterotomy

Objective: To present the feasibility of fetal myelomeningocele (MMC) repair through a mini-hysterotomy and to describe the perinatal results from our initial experience. Methods: A descriptive study of cases of fetal MMC correction via mini-hysterotomy performed between 2014 and 2016. Results: Forty-five women underwent fetal surgery and 87% (39/45) delivered. A complete multilayer correction of the MMC was possible in all cases. There were no maternal, fetal or neonatal deaths. No maternal or fetal complications occurred from fetal MMC correction until maternal hospital discharge. The average gestational age (GA) at surgery was 24.5 weeks (standard deviation, SD: 1.7; range: 20.7-26.9). The median hysterotomy length was 3.05 cm (SD: 0.39; range: 2.50-3.50). One patient (1/39; 2.6%) experienced chorioamniotic separation. Nine patients (9/39; 23.1%) had premature preterm rupture of membranes at a median GA of 34.1 weeks (range: 31.1-36.0). The average GA at delivery was 35.3 weeks (SD: 2.2; range: 27.9-39.1). Ninety-five percent (37/39) of our patients had an intact hysterotomy site at delivery. Ventriculoperitoneal shunt placement was necessary for 7.7% (3/39) of the neonates. Conclusion: Fetal MMC repair is feasible through a mini-hysterotomy. This approach appears to be associated with reduced risks of very preterm delivery and maternal, fetal and neonatal complications.

All-Arthroscopic Suprapectoral Versus Open Subpectoral Tenodesis of the Long Head of the Biceps Brachii Without the Use of Interference Screws

To compare patient-reported outcomes and healing rates after open subpectoral and all-arthroscopic suprapectoral biceps tenodesis without the use of interference screws in patients with more than 2years of follow-up. Patients with at least 2years of follow-up who underwent open subpectoral biceps tenodesis or all-arthroscopic suprapectoral biceps tenodesis without concomitant rotator cuff repair, labral repair, or Mumford procedure were considered for enrollment in the study. They were evaluated for visual analog scale (VAS), American Shoulder and Elbow Surgeons (ASES) score, and satisfaction with function and biceps contour. Ultrasonography was performed to evaluate the integrity of the tenodesis site and measure biceps muscle diameters on each arm. Forty-nine patients were eligible for our study and of these, 38 were able to participate. Twenty-three patients had open subpectoral biceps tenodesis and 15 received all-arthroscopic suprapectoral biceps tenodesis. The average follow-up time was 4.5years (range 2-9.1years). There were no significant differences in anterior shoulder pain VAS, ASES scores, or satisfaction rates. The average anterior shoulder VAS was 0.7 ± 1.1 for the open group and 0.9 ± 1.8 for the arthroscopic group (P= .74). The mean ASES score for the open group was 90.6 ± 11.4 and 91.4 ± 13.9 for the arthroscopic group (P= .69). All patients had an intact tenodesis site on ultrasonography and the ratio of operative to nonoperative biceps diameters was 100.2% ± 12.8% for the open group and 99.1% ± 10.8% for the arthroscopic group (P= .66). There were no infections and no brachial plexus injuries in either group. Open subpectoral biceps tenodesis and all-arthroscopic suprapectoral biceps tenodesis are both successful surgeries with consistently positive outcomes. Tenodesis can be performed in either location without interference screw fixation with durable, reliable results. Level III, retrospective comparative trial.

Reflecting Back to Bring Nitric Oxide Research to the Laboratory.

Professor Ferid Murad has been a remarkable colleague and a mentor. During our very first meeting, he not only shared unresolved puzzles in Nitric Oxide (NO) research but also listened to my questions pointing to protein nitration and nitrosylation. This was start of a new avenue in my laboratory involving protein nitration, inducible nitric oxide synthase and nitrite production in the context of signaling and gene expression in cancer cells. Dynamic changes in the cytoskeleton remodeling in response to the cell membrane generated signals are regulated by p21-activated kinase 1 (PAK1) which also feed into microtubules (MT) dynamic via phosphorylating Tubulin Cofactor B (CoB) on serine 65 and serine 128. While While searching for the mechanism through which MT biogenesis might be counteracted for the purpose of maintaining the balance in MT dynamic, we explored the possibility of nitration of tyrosine residues in TCoB. We found that TCoB is nitrated on tyrosine 64 and tyrosine 98 and that nitrated TCoB inhibits TCoB phosphorylation and that intact PAK1 phosphorylation sites are also essential for the ability of TCoB to undergo nitration. We suggested a model wherein TCoB nitration acts as a feedback mechanism to counteract PAK1- signaling dependent microtubule dynamics, and thus, revealed an inherent regulatory coordination of growth factor and nitric oxide signaling in microtubule dynamics. In addition, cytoskeleton remodeling and NO production and resulting post-translational modifications in signaling modules serve as important modifiers of cellular processes. Here, I will discuss the cascade of events leading to my first meeting with Professor Murad, the development of scientific interactions, the recognition of our overlapping scientific interests in NO Signaling in cancer cells, and how these interactions have allowed us to connect NO - Cytoskeleton Signaling in cancer cells.

Osteopontin Fragments with Intact Thrombin-Sensitive Site Circulate in Cervical Cancer Patients.

We investigated whether circulating osteopontin (OPN) could be used as a biomarker for cervical cancer. We employed a monoclonal antibody (mAb 659) specific for the unique and intact thrombin-sensitive site in OPN using an inhibition ELISA. We found significantly higher levels of OPN in 33 cervical cancer patients in both the plasma (mean +/- SD, 612 +/- 106 ng/mL) and serum (424 +/- 121 ng/mL) compared to healthy subjects [409 +/- 56 ng/mL, from 31 plasma samples (P < 0.0001), and 314 +/- 98 ng/mL, from 32 serum samples (P = 0.0002), respectively]. Similar results were obtained when the plasma from a bigger group (147 individuals) of cervical cancer patients (560 +/- 211 ng/mL) were compared with the same plasma samples of the healthy individuals (P = 0.0014). More significantly, the OPN level was highest in stage III-IV disease (614 +/- 210 ng/mL, from 52 individuals; P = 0.0001) and least and non-discriminatory in stage I (473 +/- 110 ng/mL, from 40 individuals; P = 0.5318). No such discrimination was found when a mAb of a different specificity (mAb 446) was used in a similar inhibition ELISA to compare the two groups in the first study; a commercial capture ELISA also failed. The possibility that the target epitope recognized by the antibody probe in these assays was absent from the circulating OPN due to protein truncation was supported by gel fractionation of the OPN found in patients’ plasma: 60–64 kDa fragments were found instead of the presumably full-length OPN (68 kDa) seen in healthy people. How these fragments are generated and what possible role they play in cancer biology remain interesting questions.

The Conserved MAPK Site in E(spl)-M8, an Effector of Drosophila Notch Signaling, Controls Repressor Activity during Eye Development.

The specification of patterned R8 photoreceptors at the onset of eye development depends on timely inhibition of Atonal (Ato) by the Enhancer of split (E(spl) repressors. Repression of Ato by E(spl)-M8 requires the kinase CK2 and is inhibited by the phosphatase PP2A. The region targeted by CK2 harbors additional conserved Ser residues, raising the prospect of regulation via multi-site phosphorylation. Here we investigate one such motif that meets the consensus for modification by MAPK, a well-known effector of Epidermal Growth Factor Receptor (EGFR) signaling. Our studies reveal an important role for the predicted MAPK site of M8 during R8 birth. Ala/Asp mutations reveal that the CK2 and MAPK sites ensure that M8 repression of Ato and the R8 fate occurs in a timely manner and at a specific stage (stage-2/3) of the morphogenetic furrow (MF). M8 repression of Ato is mitigated by halved EGFR dosage, and this effect requires an intact MAPK site. Accordingly, variants with a phosphomimetic Asp at the MAPK site exhibit earlier (inappropriate) activity against Ato even at stage-1 of the MF, where a positive feedback-loop is necessary to raise Ato levels to a threshold sufficient for the R8 fate. Analysis of deletion variants reveals that both kinase sites (CK2 and MAPK) contribute to ‘cis’-inhibition of M8. This key regulation by CK2 and MAPK is bypassed by the E(spl)D mutation encoding the truncated protein M8*, which potently inhibits Ato at stage-1 of R8 birth. We also provide evidence that PP2A likely targets the MAPK site. Thus multi-site phosphorylation controls timely onset of M8 repressor activity in the eye, a regulation that appears to be dispensable in the bristle. The high conservation of the CK2 and MAPK sites in the insect E(spl) proteins M7, M5 and Mγ, and their mammalian homologue HES6, suggest that this mode of regulation may enable E(spl)/HES proteins to orchestrate repression by distinct tissue-specific mechanisms, and is likely to have broader applicability than has been previously recognized.

Structure-function analysis and genetic interactions of the SmG, SmE, and SmF subunits of the yeast Sm protein ring.

A seven-subunit Sm protein ring forms a core scaffold of the U1, U2, U4, and U5 snRNPs that direct pre-mRNA splicing. Using human snRNP structures to guide mutagenesis in Saccharomyces cerevisiae, we gained new insights into structure–function relationships of the SmG, SmE, and SmF subunits. An alanine scan of 19 conserved amino acids of these three proteins, comprising the Sm RNA binding sites or inter-subunit interfaces, revealed that, with the exception of Arg74 in SmF, none are essential for yeast growth. Yet, for SmG, SmE, and SmF, as for many components of the yeast spliceosome, the effects of perturbing protein–RNA and protein–protein interactions are masked by built-in functional redundancies of the splicing machine. For example, tests for genetic interactions with non-Sm splicing factors showed that many benign mutations of SmG, SmE, and SmF (and of SmB and SmD3) were synthetically lethal with null alleles of U2 snRNP subunits Lea1 and Msl1. Tests of pairwise combinations of SmG, SmE, SmF, SmB, and SmD3 alleles highlighted the inherent redundancies within the Sm ring, whereby simultaneous mutations of the RNA binding sites of any two of the Sm subunits are lethal. Our results suggest that six intact RNA binding sites in the Sm ring suffice for function but five sites may not.

Testing life history and trait-based predictions of AM fungal community assembly

Arbuscular mycorrhizal (AM) fungal disturbance response is thought to be moderated by life history strategies (LHS). Research suggests that disturbance-tolerant taxa may be represented by fungi in the Glomerales, those in culture collections, and by cosmopolitan taxa due to their generalist growth habit. The corollary is that these taxa should be less common in undisturbed systems. Although widely accepted, these ideas originate from research conducted in previously disturbed systems. Whether they hold up to comparisons of disturbed versus undisturbed systems remains to be seen.We addressed this question by surveying logged and intact sites within forests dominated an AM fungal host (western redcedar; Thuja plicata). We predicted that old-growth sites would host fewer taxa from the Glomerales, fewer cultured taxa, and fewer cosmopolitan taxa compared to logged sites.Contrary to our predictions, the logged and intact sites did not differ with respect the putative disturbance-tolerant taxa. However, taxonomic composition differed, driven primarily by variation in relative abundance rather than loss or gain of taxa. Multiple analyses of indicator taxa revealed no consistent indicators of either undisturbed or disturbed habitats.Based on these findings, the current paradigm for a phylogenetically based LHS of AM fungi warrants re-examination.

Rapid Buffer Assessment Fails to Predict and Classify Wetland Floristic Quality in Oklahoma

Rapid field assessment indicators of ecological stress and disturbance may or may not reflect actual biological conditions. We tested the ability of rapidly assessed buffer quality to predict and classify wetland floristic quality across 106 sites in Oklahoma, USA. We used buffer zone metrics from a national-level rapid assessment tool (USA-RAM) to evaluate applicability at the state level. The rapid assessment relying on buffer quality did not relate predictably (linearly) with floristic quality, but the relationship improved after ecoregional stratification. Focusing on the Central Great Plains ecoregion with 55 wetlands sampled, we found 16 floristically intact sites and 13 floristically altered sites based on multivariate testing of the Floristic Quality Assessment Index and several component metrics. Distributions of buffer metrics across these sites were similar between the floristically intact and altered classes, suggesting potential for misclassification of wetland biological condition using rapid buffer assessment. Some of the disconnect may be attributed to bias in the buffer measurements and to using a national assessment protocol at the state level. Buffer metrics in the USA-RAM may offer little insight about wetland biological condition in some regions, and the use of such metrics should only complement direct bioassessments and not substitute for them.