Zinc-finger nuclease based genome surgery: it's all about specificity

Therapeutic genome engineering is a hallmark of gene therapy but only recent technological advances have permitted the modification of complex genomes in a targeted fashion. Zinc-finger nucleases (ZFNs) have developed into a major playmaker in the genome engineering field and have been employed to trigger the targeted editing of genomes at over 50 gene loci in 11 model organisms, including fruitfly, zebrafish and rat, with allelic frequencies reaching the double digit percentage range. Moreover, ZFN-mediated genome surgery in primary human cells has become a reality and two phase I clinical trials aiming at knocking out the CCR5 receptor in T cells isolated from HIV patients to protect these cells from infection with the virus have been initiated. Considering that specificity is closely linked to ZFN activity and ZFN-associated toxicity, this parameter has been and will be a key quality in any therapeutic application of the designer nucleases. This review summarizes the technological innovations that have successfully catapulted ZFNs into the genome engineering arena and provides an overview of the current state of the art of these nucleases with reference to human gene therapy.     

Diabetic vascular disease: it's all the RAGE

The major consequence of long-term diabetes is the increased incidence of disease of the vasculature. Of the underlying mechanisms leading to disease, the accumulation of advanced glycation end products (AGEs), resulting from the associated hyperglycemia, is the most convincing. Interaction of AGEs with their receptor, RAGE, activates numerous signaling pathways leading to activation of proinflammatory and procoagulatory genes. Studies in rodent models of macro- and microvascular disease have demonstrated that blockade of RAGE can prevent development of disease. These observations highlight RAGE as a therapeutic target for treatment of diabetic vascular disease.     

What is so special about thinking; after all,we all do it!

Understanding the adaptive immune system can be simplified by treating it as three linked modules, the generation of the recognitive repertoire, the sorting of the repertoire by purging anti-self, and the coupling of the repertoire to appropriate effector mechanisms. Each of these modules has a unique database and a logic that is determined by evolutionary considerations founded on value versus harm. Selection cannot operate to perfection, only to adequacy, meaning not limiting to the procreation of the species. Consequently, this system has limits in that it fails, by human standards, to adequately protect against a variety of pathogens and, even when protecting successfully against others, all too often initiates autoimmunity and innocent bystander pathology. What evolution trivializes defines the subject called clinical immunology. If we wish to deal with the pathology that evolution views to be of an acceptable frequency, then we had best first understand what it did give us as a sufficiently functional system, namely the decision pathways of the three modules and in what ways their protective outputs are limited.     

Dosing regimens: what's it all about, Alfie?

Patients on multiple drug therapies are often overwhelmed by dosing regimens for their regular drugs, and the problem becomes even more complicated when additional short-term drugs are prescribed for acute problems or opportunistic infections. A list of commonly-used medical dosing abbreviations is included (for example, b.i.d. means take twice a day). Even with this assistance, patients will have difficulty coordinating their drugs with dietary requirements. A phone number is provided for help in understanding dosing schedules.     

Protein kinase B (Akt) regulation and function in T lymphocytes

Protein kinase B (PKB) [1-5] is a serine/threonine kinase that is activated by cytokines, antigen receptors, the costimulator CD28 and chemokines in lymphocytes. [6-11] PKB is thus poised to contribute to a variety of immune activation responses. A number of functions have been ascribed to PKB in different cell lineages including the regulation of cell survival, cytokine gene induction and cell cycle progression. In the present article the mechanisms that control PKB activity in T lymphocytes will be reviewed and the function of this kinase in the immune system will be discussed.     

Genomic location of a human cytomegalovirus protein with protein kinase activity (PK68)

A major protein of Mr 68,000 with a previously described protein kinase activity (PK68), which is induced in HCMV-infected cells, is shown to be virus encoded by means of hybrid selection. The gene coding for the corresponding 4-kb mRNA was located within map units 0.510 and 0.525. This location is the same as that of pp65, a structural protein with no attributed function. The early nature of the protein was confirmed with its mRNA first appearing 3 hr after infection under cycloheximide reversal and phosphonoacetic acid blocking conditions. Another mRNA, 97 nucleotides smaller and 5' coterminal with PK68, which codes for a Mr 52,000 protein, was also observed.     

Teaching social and cultural awareness to medical students: "it's all very nice to talk about it in theory, but ultimately it makes no difference".

PURPOSE: To investigate the effect of exposure to a new course addressing social and cultural issues in medicine on third-year medical students' awareness and understanding of how these issues affect their lives as students, the lives of patients, the work of physicians, and patient-physician interaction. The course, Physicians, Patients & Society (PPS) was introduced at the time the school was moving to a PBL curriculum. METHOD: In the late 1990s, a questionnaire was administered to third-year medical students at one Canadian medical school, prior to the curriculum change (Time 1). In-depth interviews were held with 25 of these students. A few years later, the same methods were repeated (Time 2) with a third-year class that had experienced the PPS course. RESULTS: The response rate for Time 1 was 59% (n = 72), for Time 2, 51% (n = 61). Students in Time 2 did not demonstrate increased awareness of social and cultural issues. Most failed to recognize, or even denied, the effects of race, class, gender, culture, and sexual orientation. Those who acknowledged the effect of social differences tended to deny social inequality, or at best recognized disadvantages experienced by Others, but not the accompanying privileges enjoyed by their own social group. CONCLUSIONS: In general, students concluded that learning about social and cultural issues made little or no difference when they did their clinical rotations. For a medical school to produce physicians who are sensitive to and competent working with diverse communities requires a balance between attention to "difference," attention to self, and attention to power relations.     

Protein kinase C zeta suppresses low‐ or high‐grade colorectal cancer (CRC) phenotypes by interphase centrosome anchoring

Histological grading provides prognostic stratification of colorectal cancer (CRC) by scoring heterogeneous phenotypes. Features of aggressiveness include aberrant mitotic spindle configurations, chromosomal breakage, and bizarre multicellular morphology, but pathobiology is poorly understood. Protein kinase C zeta (PKCz) controls mitotic spindle dynamics, chromosome segregation, and multicellular patterns, but its role in CRC phenotype evolution remains unclear. Here, we show that PKCz couples genome segregation to multicellular morphology through control of interphase centrosome anchoring. PKCz regulates interdependent processes that control centrosome positioning. Among these, interaction between the cytoskeletal linker protein ezrin and its binding partner NHERF1 promotes the formation of a localized cue for anchoring interphase centrosomes to the cell cortex. Perturbation of these phenomena induced different outcomes in cells with single or extra centrosomes. Defective anchoring of a single centrosome promoted bipolar spindle misorientation, multi‐lumen formation, and aberrant epithelial stratification. Collectively, these disturbances induce cribriform multicellular morphology that is typical of some categories of low‐grade CRC. By contrast, defective anchoring of extra centrosomes promoted multipolar spindle formation, chromosomal instability (CIN), disruption of glandular morphology, and cell outgrowth across the extracellular matrix interface characteristic of aggressive, high‐grade CRC. Because PKCz enhances apical NHERF1 intensity in 3D epithelial cultures, we used an immunohistochemical (IHC) assay of apical NHERF1 intensity as an indirect readout of PKCz activity in translational studies. We show that apical NHERF1 IHC intensity is inversely associated with multipolar spindle frequency and high‐grade morphology in formalin‐fixed human CRC samples. To conclude, defective PKCz control of interphase centrosome anchoring may underlie distinct categories of mitotic slippage that shape the development of low‐ or high‐grade CRC phenotypes. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

DAMPs, PAMPs and alarmins: all we need to know about danger

Multicellular animals detect pathogens via a set of receptors that recognize pathogen-associated molecular patterns (PAMPs). However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Evidence is accumulating that trauma and its associated tissue damage are recognized at the cell level via receptor-mediated detection of intracellular proteins released by the dead cells. The term "alarmin" is proposed to categorize such endogenous molecules that signal tissue and cell damage. Intriguingly, effector cells of innate and adaptive immunity can secrete alarmins via nonclassical pathways and often do so when they are activated by PAMPs or other alarmins. Endogenous alarmins and exogenous PAMPs therefore convey a similar message and elicit similar responses; they can be considered subgroups of a larger set, the damage-associated molecular patterns (DAMPs).     

Protein kinase B (akt) promotes pathological angiogenesis in murine model of oxygen-induced retinopathy

Akt, or protein kinase B, is an important signaling molecule that modulates many cellular processes such as cell growth, survival, and metabolism. However, the vivo roles and effectors of Akt in retinal angiogenesis are not explicitly clear. We therefore detected the expression of Akt using Western blotting or RT-PCR technologies in an animal model of oxygen-induced retinopathy, and investigated the effects of recombinant Akt on inhibiting vessels loss and Akt inhibitor on suppressing experimental retinal neovascularization in this model. We showed that in the hyperoxic phase of oxygen-induced retinopathy, the expression of Akt was greatly suppressed. In the hypoxic phase, the expression of Akt was increased dramatically. No significant differences were found in normoxic groups. Compared with control groups, administration of the recombinant Akt in the first phase of retinopathy markedly reduced capillary-free areas, while the administration of the Akt inhibitor in the second phase of retinopathy significantly decreased retinal neovascularization but capillary-free areas. These results indicate that Akt play a critical role in the pathological process (vessels loss and neovascularization) of mouse model of oxygen-induced retinopathy, which may provide a valubale therapeutic tool for ischemic-induced retinal diseases.     

MR‐detectable metabolic consequences of mitogen‐activated protein kinase kinase (MEK) inhibition

Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS‐detectable metabolic consequences of mitogen‐activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF‐7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS‐detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF‐7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized ¹³C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3‐kinase pathway and AMP‐activated protein kinase. Our findings demonstrate the value of translatable non‐invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition. Copyright © 2014 John Wiley & Sons, Ltd.     

Andrew Fyfe the elder (1752(4)–1824): Not all good anatomists are good teachers

The history of teaching anatomy in Scotland is rich. One Scottish anatomist who has received little attention, however, is Andrew Fyfe the elder. Unfortunately, very little is written on the life and contributions of this early anatomist. He is considered to have been a great anatomist of his day, but a poor teacher of anatomy. Herein, we review the life of this early anatomist whose works have been compared to those of well‐known Scottish anatomists such as the Monros and brothers John and Charles Bell. Clin. Anat. 2013. © 2012 Wiley Periodicals, Inc.     

In Situ Forming Poly(ethylene glycol)‐ Poly(L‐lactide) Hydrogels via Michael Addition: Mechanical Properties,Degradation, and Protein Release

Chemically crosslinked hydrogels are prepared at remarkably low macromonomer concentrations from 8‐arm poly(ethylene glycol)‐poly(L ‐lactide) star block copolymers bearing acrylate end groups (PEG‐(PLLA_n)₈‐AC, n = 4 or 12) and multifunctional PEG thiols (PEG‐(SH)_n, n = 2, 4, or 8) through a Michael‐type addition reaction. Hydrogels are obtained within 1 min after mixing PEG‐(PLLA₄)₈ ‐AC and PEG‐(SH)₈ in phosphate buffered saline, quickly reaching a high storage modulus of 17 kPa. Lysozyme and albumin are released for 4 weeks from PEG‐(PLLA₁₂)₈‐AC/PEG‐(SH)₈ hydrogels. Lysozyme release from PEG‐(PLLA₁₂)₈‐AC/PEG‐(SH)₂ and PEG‐(PLLA₁₂)₈‐AC/PEG‐(SH)₄ hydrogels is significantly faster with complete release in 3 and 12 d, respectively, as a result of a combination of degradation and diffusion.