A fluorescent resonant energy transfer–based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases

Development of a Scalable Pharmacogenomic Clinical Decision Support Service

Advances in sequencing technology are making genomic data more accessible within the healthcare environment. Published pharmacogenetic guidelines attempt to provide a clinical context for specific genomic variants; however, the actual implementation to convert genomic data into a clinical report integrated within an electronic medical record system is a major challenge for any hospital. We created a two-part solution that integrates with the medical record system and converts genetic variant results into an interpreted clinical report based on published guidelines. We successfully developed a scalable infrastructure to support TPMT genetic testing and are currently testing approximately two individuals per week in our production version. We plan to release an online variant to clinical interpretation reporting system in order to facilitate translation of pharmacogenetic information into clinical practice

Peripheral nerve growth within a hydrogel microchannel scaffold supported by a kink‐resistant conduit

Nerve repair in several mm‐long nerve gaps often requires an interventional technology. Microchannel scaffolds have proven effective for bridging nerve gaps and guiding axons in the peripheral nervous system (PNS). Nonetheless, fabricating microchannel scaffolds at this length scale remains a challenge and/or is time consuming and cumbersome. In this work, a simple computer‐aided microdrilling technique was used to fabricate 10 mm‐long agarose scaffolds consisting of 300 µm‐microchannels and 85 µm‐thick walls in less than an hour. The agarose scaffolds alone, however, did not exhibit adequate stiffness and integrity to withstand the mechanical stresses during implantation and suturing. To provide mechanical support and enable suturing, poly caprolactone (PCL) conduits were fabricated and agarose scaffolds were placed inside. A modified salt‐leaching technique was developed to introduce interconnected porosity in PCL conduits to allow for tuning of the mechanical properties such as elastic modulus and strain to failure. It was shown that the PCL conduits were effective in stabilizing the agarose scaffolds in 10 mm‐long sciatic nerve gaps of rats for at least 8 weeks. Robust axon ingress and Schwann cell penetration were observed within the microchannel scaffolds without using growth factors. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3392–3399, 2017.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/139110/1/jbma36186_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/139110/2/jbma36186.pd

HIV-1 Phenotypic Reverse Transcriptase Inhibitor Drug Resistance Test Interpretation Is Not Dependent on the Subtype of the Virus Backbone

To date, the majority of HIV-1 phenotypic resistance testing has been performed with subtype B virus backbones (e.g. HXB2). However, the relevance of using this backbone to determine resistance in non-subtype B HIV-1 viruses still needs to be assessed. From 114 HIV-1 subtype C clinical samples (36 ARV-naïve, 78 ARV-exposed), pol amplicons were produced and analyzed for phenotypic resistance using both a subtype B- and C-backbone in which the pol fragment was deleted. Phenotypic resistance was assessed in resulting recombinant virus stocks (RVS) for a series of antiretroviral drugs (ARV's) and expressed as fold change (FC), yielding 1660 FC comparisons. These Antivirogram® derived FC values were categorized as having resistant or sensitive susceptibility based on biological cut-off values (BCOs). The concordance between resistance calls obtained for the same clinical sample but derived from two different backbones (i.e. B and C) accounted for 86.1% (1429/1660) of the FC comparisons. However, when taking the assay variability into account, 95.8% (1590/1660) of the phenotypic data could be considered as being concordant with respect to their resistance call. No difference in the capacity to detect resistance associated with M184V, K103N and V106M mutations was noted between the two backbones. The following was concluded: (i) A high level of concordance was shown between the two backbone phenotypic resistance profiles; (ii) Assay variability is largely responsible for discordant results (i.e. for FC values close to BCO); (iii) Confidence intervals should be given around the BCO's, when assessing resistance in HIV-1 subtype C; (iv) No systematic resistance under- or overcalling of subtype C amplicons in the B-backbone was observed; (v) Virus backbone subtype sequence variability outside the pol region does not contribute to phenotypic FC values. In conclusion the HXB2 virus backbone remains an acceptable vector for phenotyping HIV-1 subtype C pol amplicons

A Hox-Embedded Long Noncoding RNA: Is It All Hot Air?

Over 20 years ago, the discovery of Xist as a critical component of X chromosome inactivation revealed a fundamental role for long noncoding RNAs (lncRNAs) in epigenetic regulation during mammalian development and foreshadowed a fascinating connection between RNA and chromatin modification [1–3]. In the last decade, the field has exploded, heralded in part by a 2007 landmark paper from the group of Howard Chang [4] describing that knockdown of a lncRNA (Hox Antisense Intergenic RNA [HOTAIR]) was associated with loss of transcriptional repression from a locus on another chromosome in trans. HOTAIR lncRNA—encoded within the HOXC locus, although its expression seemed to be required for normal epigenetic silencing of HOXD genes—became one of the most well-known examples of functional lncRNAs in the field of developmental epigenetics. Interest intensified when a subsequent paper from the Chang lab [5] reported that targeted deletion of the orthologous locus in the mouse (Hotair) caused homeotic transformations underpinned by derepression of HoxD gene transcription in vivo. Discovery of new lncRNAs and exploration of their potential actions and effects during development and disease is a continued source of excitement [6,7]. But questions about the effects and actions of Hotair have been controversial, raised in part by work from the group of Denis Duboule [8]. This debate is addressed directly in the current issue of PLOS Genetics in a manuscript from Duboule’s laboratory [9] that reanalyzes Hotair mutant mice generated by the Chang lab, a formal comment in response to that manuscript from the Chang lab [10], and this perspective

5-Formylcytosine organizes nucleosomes and forms Schiff base interactions with histones in mouse embryonic stem cells.

Nucleosomes are the basic unit of chromatin that help the packaging of genetic material while controlling access to the genetic information. The underlying DNA sequence, together with transcription-associated proteins and chromatin remodelling complexes, are important factors that influence the organization of nucleosomes. Here, we show that the naturally occurring DNA modification, 5-formylcytosine (5fC) is linked to tissue-specific nucleosome organization. Our study reveals that 5fC is associated with increased nucleosome occupancy in vitro and in vivo. We demonstrate that 5fC-associated nucleosomes at enhancers in the mammalian hindbrain and heart are linked to elevated gene expression. Our study also reveals the formation of a reversible-covalent Schiff base linkage between lysines of histone proteins and 5fC within nucleosomes in a cellular environment. We define their specific genomic loci in mouse embryonic stem cells and look into the biological consequences of these DNA-histone Schiff base sites. Collectively, our findings show that 5fC is a determinant of nucleosome organization and plays a role in establishing distinct regulatory regions that control transcription

Higher carbohydrate intake is associated with increased risk of allâ cause and diseaseâ specific mortality in head and neck cancer patients: results from a prospective cohort study

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145268/1/ijc31413.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145268/2/ijc31413-sup-0001-suppinfo01.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145268/3/ijc31413_am.pd