Multi-Organ Expression Profiling Uncovers a Gene Module in Coronary Artery Disease Involving Transendothelial Migration of Leukocytes and LIM Domain Binding 2: The Stockholm Atherosclerosis Gene Expression (STAGE) Study

Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10−27and−30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2–deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research

A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry

Transcriptional Regulation by CHIP/LDB Complexes

It is increasingly clear that transcription factors play versatile roles in turning genes “on” or “off” depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. Here we use the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, we identified 189 down-stream targets of CHIP/LDB and show that these genes are enriched for the binding sites of APTEROUS (AP) and PANNIER (PNR), two well studied transcription factors associated with CHIP/LDB complexes. We performed extensive genetic screens and identified target genes that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing we uncovered novel roles for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, our results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development

Analysis of the DNA polymerase promoter of human herpesvirus-6

Human herpesvirus-6 (HHV-6) is a recently described T-cell pathogen whose medical relevance and molecular biology are just beginning to be addressed. As a first look at the regulation of viral genes, control of the HHV-G DNA polymerase promoter was examined. Polymerase gene transcription in HHV-6 infected cells was found to initiate from a single site located 115 bases upstream of the translation start codon. A polymerase promoter-chloramphenicol acetyltransferase (CAT) reporter gene construct failed to be expressed in uninfected T-cells but was highly active in HHV-6 infected cells. Mutational data indicated that the polymerase promoter is TATA-less. Mutational analysis also revealed that the major upstream promoter regulatory element required for transcriptional activity in HHV-6 infected cells is a palindromic ATF/CREB transcription factor binding site. An adjacent purine box does not appear to contribute significantly to activity. The significance of the ATF/CREB site for promoter induction was further demonstrated by the fact that the polymerase ATF/CREB element, when appended to a heterologous basal promoter, is highly responsive to HHV-6 infection. Two protein complexes were found to bind in a specific manner to the ATF/CREB motif in both uninfected and HHV-6 infected T-cell nuclear extracts. One of these complexes was shown to contain ATF-2 or an ATF-2 like protein. Site specific mutation of the ATF/CREB site resulted in loss of protein binding as well as loss of promoter activity in HHV-6 infected cells, suggesting a requirement for protein binding for promoter function. Multiple specific protein complexes were found to bind to the purine box motif in both uninfected and infected cells. A cDNA encoding a 44 kd protein which binds to the purine box was cloned

Why are spine surgeons sued, and with what outcomes?

Background: Why are spine surgeons sued, how successfully, and for how much? Typical bases for spinal medicolegal suits have included; the failure to timely diagnose and treat, surgical negligence, (i.e. especially resulting in significant neurological deficits), and the lack of informed consent. We reviewed 17 medicolegal spinal articles looking for additional reasons for suits, along with identifying other factors contributing to defense verdicts, plaintiffs’ verdicts, or settlements. Methods: After confirming the same three most likely causes of medicolegal suits, other factors leading to such suits included; the lack of patient access to surgeons postoperatively, poor postoperative management (i.e. contributing to new postoperative neurological deficits), failure to communicate between specialists/surgeons perioperatively, and failure to brace. Results: Critical factors leading to more plaintiffs’ verdicts and settlements along with higher payouts for both included new severe and/or catastrophic postoperative neurological deficits. Conversely, defense verdicts were more likely for those with less severe new and/or residual injuries. The total number of plaintiffs’ verdicts ranged from 17-35.2%, settlements, from 8.3-37%, and defense verdicts from 27.7-75%. Conclusion: The three most frequent bases for spinal medicolegal suits continue to include; failure to timely diagnose/treat, surgical negligence, and lack of informed consent. Here, we identified the following additional causes of such suits; the lack of patient access to surgeons perioperatively, poor postoperative management, lack of specialist/surgeon communication, and failure to brace. Further, more plaintiffs’ verdicts or settlements and greater respective payouts were observed for those with new and/or more severe/catastrophic deficits, while more defense verdicts were typically rendered for patients with lesser new neurological injuries. </jats:sec

Reconstruction of Shattered Lumbo-Sacral Junction/Pelvis Utilizing Bilateral L4-Sacrum Fibula Strut Allograft And Double Iliac Screws Plus Routine Lumbar Pedicle Screw Fixation

Background: A traumatically shattered lumbosacral junction/pelvis may be difficult to repair. Here the authors offer a pelvic fixation technique utilizing routine pedicle screws, interbody lumbar fusions, bilateral iliac screws/ rods/crosslinks, and bilateral fibular strut allografts from the lumbar spine to the sacrum. Methods: A middle aged male sustained a multiple storey fall resulting in a left sacral fracture, and right sacroiliac joint (SI) dislocation. The patient had previously undergone attempted decompressions with routine pedicle screw L4-S1 fusions at outside institutions; these failed twice. When the patient was finally seen, he exhibited, on CT reconstructed images, MR, and X-rays, a left sacral fracture nonunion, and a right sacroiliac joint dislocation. Results: The patient underwent a bilateral pelvic reconstruction utilizing right L4, L5, S1 and left L4, L5 pedicle screws plus interbody fusions (L4-L5, and L5, S1), performed from the left. Unique to this fusion construct was the placement of bilateral double iliac screws plus the application of bilateral fibula allografts from L4-sacrum filled with bone morphogenetic protein (BMP). After rod/screw/connectors were applied, bone graft was placed over the fusion construct, including the decorticated edges of the left sacral fractures, and right SI joint dislocation. We additionally reviewed other pelvic fusion reconstruction methods. Conclusions: Here, we utilized a unique pelvic reconstruction technique utilizing pedicle screws/rods, double iliac screws/rods, and bilateral fibula strut grafts extending from the L4-sacrum filled with BMP. </jats:sec

Distinct Representations of Subtraction and Multiplication in the Neural Systems for Numerosity and Language - A replication study

A core paradox within cognitive science is the emergence of cultural functions, such as writing systems and arithmetic, that develop across time spans far too short for our neural systems to evolve to support them. Previous work has addressed this question with the neural recycling hypothesis, proposing that these newer functions are mapped onto pre-existing interconnected regions of the brain, called neural circuits. We replicated results from a study exploring the specific functions that have been recycled to allow for symbolic subtraction and multiplication. Original findings suggested that numerosity circuitry, typically responsible for comparing the size or quantity of two groups, is employed for subtraction and verbal processing circuitry for multiplication. We reviewed the collected fMRI data to construct a model of the brain with the region responsible for numerosity localized. We confirmed that the region localized by the numerosity task corresponded to the right intraparietal sulcus (IPS). Future research should focus on the corollary of the neural circuitry hypothesis—that later-evolving processes are subject to the restrictions of the circuitry they recycle. In particular, analyzing data obtained from incorrect answers to tasks would help confirm that recycling underlies the correlations we see between the neural activity of certain tasks