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Microscope basics

Wed, 06/11/2014 - 10:05am

This chapter provides information on how microscopes work and discusses some of the microscope issues to be considered in using a video camera on the microscope. There are two types of microscopes in use today for research in cell biology-the older finite tube-length (typically 160mm mechanical tube length) microscopes and the infinity optics microscopes that are now produced. The objective lens forms a magnified, real image of the specimen at a specific distance from the objective known as the intermediate image plane. All objectives are designed to be used with the specimen at a defined distance from the front lens element of the objective (the working distance) so that the image formed is located at a specific location in the microscope. Infinity optics microscopes differ from the finite tube-length microscopes in that the objectives are designed to project the image of the specimen to infinity and do not, on their own, form a real image of the specimen. Three types of objectives are in common use today-plan achromats, plan apochromats, and plan fluorite lenses. The concept of mounting video cameras on the microscope is also presented in the chapter.

Practical aspects of adjusting digital cameras

Wed, 06/11/2014 - 10:05am

This chapter introduces the adjustment of digital camera settings using the tools found within image acquisition software and discusses measuring gray-level information such as (1) the histogram, (2) line scan, and (3) other strategies. The pixel values in an image can be measured within many image capture software programs in two ways. The first is a histogram of pixel gray values and the second is a line-scan plot across a selectable axis of the image. Understanding how to evaluate the information presented by these tools is critical to properly adjusting the camera to maximize the image contrast without losing grayscale information. This chapter discusses the 0-255 grayscale resolution of an 8-bit camera; however, the concepts are the same for cameras of any bit depth. This chapter also describes camera settings, such as exposure time, offset, and gain, and the steps for contrast stretching such as setting the exposure time, adjusting offset and gain, and camera versus image display controls.

Preface. Digital microscopy

Wed, 06/11/2014 - 10:05am

Centriole engagement: it's not just cohesin any more

Wed, 06/11/2014 - 10:05am

The belief that cohesin complexes link mother to daughter centrioles has received substantial experimental support. New studies challenge the primacy of cohesin in centriole engagement and provide a more nuanced view into the mechanisms for centriole disengagement in anaphase.

Link Between DNA Damage and Centriole Disengagement/Reduplication in Untransformed Human Cells

Wed, 06/11/2014 - 10:05am

The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 hours, though 14% of the cells eventually go through mitosis in that time. By 72 hours after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with gamma-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53 dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. J. Cell. Physiol. (c) 2014 Wiley Periodicals, Inc.

NrasG12D oncoprotein inhibits apoptosis of pre-leukemic cells expressing Cbfbeta-SMMHC via activation of MEK/ERK axis

Tue, 06/10/2014 - 11:09am

Acute myeloid leukemia (AML) results from the activity of driver mutations that deregulate proliferation and survival of hematopoietic stem cells (HSCs). The fusion protein CBFbeta-SMMHC impairs differentiation in hematopoietic stem and progenitor cells, and induces AML in cooperation with other mutations. However, the combined function of CBFbeta-SMMHC and cooperating mutations in pre-leukemic expansion is not known. Here, we used NrasLSL-G12D; Cbfb56M knock-in mice to show that allelic expression of oncogenic NrasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic short-term HSCs and myeloid progenitor cells and maintains the differentiation block induced by the fusion protein. NrasG12D and Cbfbeta-SMMHC synergize to induce leukemia in mice in a cell autonomous manner with a median latency shorter and with higher leukemia-initiating cell activity than that of mice expressing Cbfbeta-SMMHC. Furthermore, NrasLSL-G12D; Cbfb56M leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway, increasing apoptosis and Bim protein levels. These studies demonstrate that Cbfbeta-SMMHC and NrasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv16 AML targeted therapies.

Cancer-Relevant Splicing Factor CAPERalpha Engages the Essential Splicing Factor SF3b155 in a Specific Ternary Complex

Tue, 06/10/2014 - 11:09am

U2AF Homology Motifs (UHMs) mediate protein-protein interactions with U2AF Ligand Motifs (ULMs) of pre-mRNA splicing factors. The UHM-containing alternative splicing factor CAPERalpha regulates splicing of tumor-promoting VEGF isoforms, yet the molecular target of the CAPERalpha UHM is unknown. Here, we present structures of the CAPERalpha UHM bound to a representative SF3b155 ULM at 1.7 A resolution, and for comparison, in the absence of ligand at 2.2 A resolution. The prototypical UHM/ULM interactions authenticate CAPERalpha as a bona fide member of the UHM-family of proteins. We identify SF3b155 as the relevant ULM-containing partner of full-length CAPERalpha in human cell extracts. Isothermal titration calorimetry comparisons of the purified CAPERalpha UHM binding known ULM-containing proteins demonstrate that high affinity interactions depend on the presence of an intact, intrinsically-unstructured SF3b155 domain containing seven ULM-like motifs. The interplay among bound CAPERalpha molecules gives rise to the appearance of two high affinity sites in the SF3b155 ULM-containing domain. In conjunction with the previously-identified, UHM/ULM-mediated complexes of U2AF65 and SPF45 with SF3b155, this work demonstrates the capacity of SF3b155 to offer a platform for coordinated recruitment of UHM-containing splicing factors.

Transcriptional regulation of Caenorhabditis elegans FOXO/DAF-16 modulates lifespan

Tue, 06/10/2014 - 11:09am

BACKGROUND: Insulin/IGF-1 signaling plays a central role in longevity across phylogeny. In C. elegans, the forkhead box O (FOXO) transcription factor, DAF-16, is the primary target of insulin/IGF-1 signaling, and multiple isoforms of DAF-16 (a, b, and d/f) modulate lifespan, metabolism, dauer formation, and stress resistance. Thus far, across phylogeny modulation of mammalian FOXOs and DAF-16 have focused on post-translational regulation with little focus on transcriptional regulation. In C. elegans, we have previously shown that DAF-16d/f cooperates with DAF-16a to promote longevity. In this study, we generated transgenic strains expressing near-endogenous levels of either daf-16a or daf-16d/f, and examined temporal expression of the isoforms to further define how these isoforms contribute to lifespan regulation. RESULTS: Here, we show that DAF-16a is sensitive both to changes in gene dosage and to alterations in the level of insulin/IGF-1 signaling. Interestingly, we find that as worms age, the intestinal expression of daf-16d/f but not daf-16a is dramatically upregulated at the level of transcription. Preventing this transcriptional upregulation shortens lifespan, indicating that transcriptional regulation of daf-16d/f promotes longevity. In an RNAi screen of transcriptional regulators, we identify elt-2 (GATA transcription factor) and swsn-1 (core subunit of SWI/SNF complex) as key modulators of daf-16d/f gene expression. ELT-2 and another GATA factor, ELT-4, promote longevity via both DAF-16a and DAF-16d/f while the components of SWI/SNF complex promote longevity specifically via DAF-16d/f. CONCLUSIONS: Our findings indicate that transcriptional control of C. elegans FOXO/daf-16 is an essential regulatory event. Considering the conservation of FOXO across species, our findings identify a new layer of FOXO regulation as a potential determinant of mammalian longevity and age-related diseases such as cancer and diabetes.

RLIM is dispensable for X-chromosome inactivation in the mouse embryonic epiblast

Tue, 06/10/2014 - 11:09am

In female mice, two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X chromosome. Later, around implantation, epiblast cells of the inner cell mass that give rise to the embryo reactivate the paternal X chromosome and undergo a random form of XCI (rXCI). Xist, a long non-coding RNA crucial for both forms of XCI, is activated by the ubiquitin ligase RLIM (also known as Rnf12). Although RLIM is required for triggering iXCI in mice, its importance for rXCI has been controversial. Here we show that RLIM levels are downregulated in embryonic cells undergoing rXCI. Using mouse genetics we demonstrate that female cells lacking RLIM from pre-implantation stages onwards show hallmarks of XCI, including Xist clouds and H3K27me3 foci, and have full embryogenic potential. These results provide evidence that RLIM is dispensable for rXCI, indicating that in mice an RLIM-independent mechanism activates Xist in the embryo proper.

Estrogen defines the dorsal-ventral limit of VEGF regulation to specify the location of the hemogenic endothelial niche

Tue, 06/10/2014 - 11:09am

Genetic control of hematopoietic stem and progenitor cell (HSPC) function is increasingly understood; however, less is known about the interactions specifying the embryonic hematopoietic niche. Here, we report that 17beta-estradiol (E2) influences production of runx1+ HSPCs in the AGM region by antagonizing VEGF signaling and subsequent assignment of hemogenic endothelial (HE) identity. Exposure to exogenous E2 during vascular niche development significantly disrupted flk1+ vessel maturation, ephrinB2+ arterial identity, and specification of scl+ HE by decreasing expression of VEGFAa and downstream arterial Notch-pathway components; heat shock induction of VEGFAa/Notch rescued E2-mediated hematovascular defects. Conversely, repression of endogenous E2 activity increased somitic VEGF expression and vascular target regulation, shifting assignment of arterial/venous fate and HE localization; blocking E2 signaling allowed venous production of scl+/runx1+ cells, independent of arterial identity acquisition. Together, these data suggest that yolk-derived E2 sets the ventral boundary of hemogenic vascular niche specification by antagonizing the dorsal-ventral regulatory limits of VEGF.

Surgical Complications and Emergencies

Mon, 06/09/2014 - 10:25am

Book chapter describing complications and emergencies that might arise during dermatological surgery.

Surgical Dermatopathology

Mon, 06/09/2014 - 10:25am

Publisher description: Surgical dermatology is a growing field reflecting the increasing incidence of photodamage and sun-related tumors, whether benign or malignant. These diseases require expert pathological diagnosis -- sometimes interoperatively -- and therefore all it is important to learn not only techniques of lesion removal, but also specimen preparation and histological appearance. Surgical dermatology covers the gamut of surgical skin lesions, clinical features, histologic subtypes, frozen section and permanent section interpretation, and differential diagnosis. Normal variants are covered in depth. Laboratory techniques for frozen, permanent and MOHS fixed tissue preparation are given. There is a very useful section on artifacts and margin control, both of which have important practical implications. Because other dermatopathology texts cover all dermatologic diseases, they do not focus in such depth on the particular problems of technique and recognition proposed by dermatosurgical specimens.

Cutaneous Oncology: Pathophysiology, Diagnosis, and Management

Mon, 06/09/2014 - 10:25am

Publisher description: This book was written to fill a perceived void in the current literature. Cutaneous Oncology is designed to focus on the nature and behaviour of cancers, standing at the junction of clinical medicine and basic science research to provide a whole picture of skin tumor biology to both clinicians and researchers. The book explains our clinical understanding of skin cancers in terms of their biological and molecular bases, and provides a framework of clinical settings in which to understand and appreciate the importance of individual facets of tumor biology.

Textbook of Dermatologic Surgery

Mon, 06/09/2014 - 10:25am

Publisher description: This volume is a comprehensive and highly practical textbook of dermatologic surgery. The book provides clear, detailed, step-by-step how-to instructions for all available surgical procedures and techniques and offers precise guidance on recognizing pitfalls, avoiding complications, and managing them when they occur. Close attention is also given to postoperative management. More than 1,200 detail-revealing illustrations complement the text throughout. Coverage encompasses all procedures using steel instruments and lasers and special procedures such as Mohs surgery, cryosurgery, electrosurgery, dermabrasion, chemical peels, hair replacement, suction lipectomy, augmentation, and sclerotherapy. For each procedure, the text discusses preoperative planning; technique, with anatomic considerations if applicable; postoperative management; and complications.

Surgical Complications

Mon, 06/09/2014 - 10:25am

Infection Control

Mon, 06/09/2014 - 10:25am

Basic Skin Surgery Techniques

Mon, 06/09/2014 - 10:25am

Differential Muscle Hypertrophy Is Associated with Satellite Cell Numbers and Akt Pathway Activation Following Activin Type IIB Receptor Inhibition in Mtm1 p.R69C Mice

Mon, 06/09/2014 - 8:54am

X-linked myotubular myopathy is a congenital myopathy caused by deficiency of myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-linked myotubular myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence of hypertrophy in gastrocnemius muscles but not in quadriceps or triceps. The ability of the muscles to respond to activin receptor type IIB inhibitor treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. Treatment-responsive Mtm1 p.R69C gastrocnemius muscles displayed lower levels of phosphorylated ribosomal protein S6 and higher levels of phosphorylated eukaryotic elongation factor 2 kinase than were observed in Mtm1 p.R69C quadriceps muscle or in muscles from wild-type littermates. Hypertrophy in the Mtm1 p.R69C gastrocnemius muscle was associated with increased levels of phosphorylated ribosomal protein S6. Our findings indicate that muscle-, fiber type-, and mutation-specific factors affect the response to hypertrophic therapies that will be important to assess in future therapeutic trials.

Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death

Thu, 06/05/2014 - 3:15pm

A number of pathogens cause host cell death upon infection, and Yersinia pestis, infamous for its role in large pandemics such as the "Black Death" in medieval Europe, induces considerable cytotoxicity. The rapid killing of macrophages induced by Y. pestis, dependent upon type III secretion system effector Yersinia outer protein J (YopJ), is minimally affected by the absence of caspase-1, caspase-11, Fas ligand, and TNF. Caspase-8 is known to mediate apoptotic death in response to infection with several viruses and to regulate programmed necrosis (necroptosis), but its role in bacterially induced cell death is poorly understood. Here we provide genetic evidence for a receptor-interacting protein (RIP) kinase-caspase-8-dependent macrophage apoptotic death pathway after infection with Y. pestis, influenced by Toll-like receptor 4-TIR-domain-containing adapter-inducing interferon-β (TLR4-TRIF). Interestingly, macrophages lacking either RIP1, or caspase-8 and RIP3, also had reduced infection-induced production of IL-1β, IL-18, TNF, and IL-6; impaired activation of the transcription factor NF-κB; and greatly compromised caspase-1 processing. Cleavage of the proform of caspase-1 is associated with triggering inflammasome activity, which leads to the maturation of IL-1β and IL-18, cytokines important to host responses against Y. pestis and many other infectious agents. Our results identify a RIP1-caspase-8/RIP3-dependent caspase-1 activation pathway after Y. pestis challenge. Mice defective in caspase-8 and RIP3 were also highly susceptible to infection and displayed reduced proinflammatory cytokines and myeloid cell death. We propose that caspase-8 and the RIP kinases are key regulators of macrophage cell death, NF-κB and inflammasome activation, and host resistance after Y. pestis infection.

Understanding the Last Days of Life: Information and Resources for Patients and Family Members

Thu, 06/05/2014 - 3:04pm

The purpose of this article is to describe my experience during the last few days of my father's life. My father had been suffering with dementia for approximately eight years. As a family, we worked together to plan his care. However, we were not prepared for his last days of life. Understanding the process helped me cope with the end of my father's life. The end of the article includes links to some freely available resources to help you, your family, and your patrons.