Phosphorylation of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A circuit

In higher eukaryotic organisms, the checkpoint kinase 1 (Chk1) contributes essential functions to both cell cycle and checkpoint control. Chk1 executes these functions, in part, by targeting the Cdc25A protein phosphatase for ubiquitin-mediated proteolysis. In response to genotoxic stress, Chk1 is phosphorylated on serines 317 (S317) and 345 (S345) by the ataxia-telangiectasia-related (ATR) protein kinase. Phosphorylation of Chk1 on these C-terminal serine residues is used as an indicator of Chk1 activation in vivo. Here, we report that inhibition of Chk1 kinase activity paradoxically leads to the accumulation of S317- and S345-phosphorylated Chk1 in vivo and that ATR catalyzes Chk1 phosphorylation under these conditions. We demonstrate that Chk1 phosphorylation by ATR is antagonized by protein phosphatase 2A (PP2A). Importantly, dephosphorylation of Chk1 by PP2A is regulated, in part, by the kinase activity of Chk1. We propose that the ATR-Chk1-PP2A regulatory circuit functions to keep Chk1 in a low-activity state during an unperturbed cell division cycle but at the same time keeps Chk1 primed to respond rapidly in the event that cells encounter genotoxic stress.     

Cancer Genetics Education in a Low- to Middle-Income Country: Evaluation of an Interactive Workshop for Clinicians in Kenya

Background

Clinical genetic testing is becoming an integral part of medical care for inherited disorders. While genetic testing and counseling are readily available in high-income countries, in low- and middle-income countries like Kenya genetic testing is limited and genetic counseling is virtually non-existent. Genetic testing is likely to become widespread in Kenya within the next decade, yet there has not been a concomitant increase in genetic counseling resources. To address this gap, we designed an interactive workshop for clinicians in Kenya focused on the genetics of the childhood eye cancer retinoblastoma. The objectives were to increase retinoblastoma genetics knowledge, build genetic counseling skills and increase confidence in those skills.

Methods

The workshop was conducted at the 2013 Kenyan National Retinoblastoma Strategy meeting. It included a retinoblastoma genetics presentation, small group discussion of case studies and genetic counseling role-play. Knowledge was assessed by standardized test, and genetic counseling skills and confidence by questionnaire.

Results

Knowledge increased significantly post-workshop, driven by increased knowledge of retinoblastoma causative genetics. One-year post-workshop, participant knowledge had returned to baseline, indicating that knowledge retention requires more frequent reinforcement. Participants reported feeling more confident discussing genetics with patients, and had integrated more genetic counseling into patient interactions.

Conclusion

A comprehensive retinoblastoma genetics workshop can increase the knowledge and skills necessary for effective retinoblastoma genetic counseling.     

Genetic mutation analysis at early stages of cell line development using next generation sequencing

A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. However, one quality attribute has, until recently, been absent from the standard battery of analytical tests facilitating informed choices early in cell line selection: genetic sequence confirmation. Techniques historically used for mutation analysis, such as detailed mass spectrometry, have limitations on the sample number and turnaround times making it less attractive at early stages. Thus, we explored the utility of Next‐Generation Sequencing (NGS) as a solution to address these limitations. Amplicon sequencing is one such NGS technique that is robust, rapid, sensitive, and amenable to multiplexing, all of which are essential attributes for our purposes. Here we report a NGS method based upon amplicon sequencing that has been successfully incorporated into our cell line development workflow alongside other high‐throughput protein analytical assays. The NGS method has demonstrated its value by identifying at least one Chinese hamster ovary (CHO) clone expressing a variant form of the biotherapeutic in each of the four clinical programs in which it has been utilized. We believe this sequence confirmation method is essential to safely accelerating the time to clinical proof of concept of biotherapeutics, and guard against delays related to sequence mutations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:813–817, 2016     

Enhanced branching morphogenesis in mammary glands of mice lacking cell surface beta1,4-galactosyltransferase

Development of the mammary gland is influenced both by the systemic hormonal environment and locally through cell-cell and cell-extracellular matrix (ECM) interactions. We have previously demonstrated aberrant mammary gland morphogenesis in transgenic mice with elevated levels of the long isoform of beta1,4-galactosyltransferase 1 (GalT), a proportion of which is targeted to the plasma membrane, where it plays a role in cell-ECM interactions. Here, we show that mammary glands of mice lacking the long GalT isoform exhibit a complementary phenotype. Cell-surface GalT activity was reduced by over 60%, but because the short GalT isoform is intact, total GalT activity was reduced only slightly relative to wild type. Mammary glands from long GalT-null mice were characterized by excess branching, and this phenotype was accompanied by altered expression of laminin chains. Laminin alpha1 and alpha3 were reduced 2.4- and 3.0-fold, respectively, while expression of laminin gamma2 was elevated 2.3-fold. The expression and cleavage of laminin gamma2 have been correlated with branching and cell migration, and Western blotting revealed an altered pattern in gamma2 cleavage products in long GalT-null mammary glands. We then examined the expression of metalloproteases that cleave laminins or that have been shown to play a role in mammary gland morphogenesis. Expression of MT1-MMP, a membrane-bound protease that can cleave laminin gamma2, was elevated 5.5-fold in the long GalT-nulls. MMP 7 was also elevated 5.1-fold. Our results suggest that expression of surface GalT is important for the proper regulation of matrix expression and deposition, which in turn regulates the proper branching morphogenesis of the mammary epithelial ductal system.     

Comparison of vitrification and slow cooling for umbilical tissues

The tissue cryopreservation maintains the cellular metabolism in a quiescence state and makes the conservation possible for an indefinite period of time. The choice of an appropriate cryopreservation protocol is essential for maintenance of cryopreserved tissue banks. This study evaluated 10 samples of umbilical cord, from which small fragments of tissue (Wharton’s jelly and cord lining membrane) were subjected to two protocols of cryopreservation: slow cooling and vitrification. The samples were frozen for a period of time ranging from 5 to 78 days. The efficiency of cryopreservation was evaluated by testing cell viability, histological analysis, cell culture, cytogenetic analysis and comparison with the results of the fresh samples. The results showed that the slow cooling protocol was more efficient than the vitrification for cryopreservation of umbilical cord tissue, because it has caused fewer changes in the structure of tissue (edema and degeneration of the epithelium) and, despite the significant decrease cell viability compared to fresh samples, the ability of cell proliferation in vitro was preserved in most samples. In conclusion, this study showed that it is possible to cryopreserve small fragments of tissue from the umbilical cord and, to obtain viable cells capable of proliferation in vitro after thawing, contributing to the creation of a frozen tissue bank.     

Overcoming Efficiency Limitations of SnS‐Based Solar Cells

Thin‐film solar cells are made by vapor deposition of Earth‐abundant materials: tin, zinc, oxygen and sulfur. These solar cells had previously achieved an efficiency of about 2%, less than 1/10 of their theoretical potential. Loss mechanisms are systematically investigated and mitigated in solar cells based on p‐type tin monosulfide, SnS, absorber layers combined with n‐type zinc oxysulfide, Zn(O,S) layers that selectively transmit electrons, but block holes. Recombination at grain boundaries is reduced by annealing the SnS films in H₂S to form larger grains with fewer grain boundaries. Recombination near the p‐SnS/n‐Zn(O,S) junction is reduced by inserting a few monolayers of SnO₂ between these layers. Recombination at the junction is also reduced by adjusting the conduction band offset by tuning the composition of the Zn(O,S), and by reducing its free electron concentration with nitrogen doping. The resulting cells have an efficiency over 4.4%, which is more than twice as large as the highest efficiency obtained previously by solar cells using SnS absorber layers.     

Inbreeding depression and selfing rate of Ipomoea hederacea var. integriuscula (Convolvulaceae)

Inbreeding depression and selfing rate were investigated in the self-compatible vine Ipomoea hederacea to assess the variability of the breeding system. Inbreeding depression differed between populations and the magnitude varied at germination, growth (as measured by aboveground biomass), and reproductive potential. Plants from Macon County, Alabama, USA, had significant inbreeding depression (31%) at germination, but no significant inbreeding depression for aboveground biomass or number of reproductive structures (buds and flowers) at 45 d post germination in the greenhouse or in the field. Plants from Morgan County, Alabama, however, had significant inbreeding depression (>50%) for all three stages in the greenhouse. In allozyme comparisons, five of the 11 I. hederacea populations surveyed had high selfing rates (66.66-92.53%) and high levels of homozygosity (F(IS) = 0.500-0.861) in 2003, and three of four populations surveyed in 2004 had selfing rates that exceeded 50%. High selfing rates, high levels of homozygosity, and low levels of inbreeding depression suggest that inbreeding depression may not present a significant barrier to the transmission of selfing alleles in some populations of I. hederacea, but does not account for the maintenance of a mixed mating system in other populations.     

Melanin-concentrating hormone and melanin-concentrating hormone receptors in mammalian skin physiopathology

To date, there is a dearth of evidence to support functions for melanin-concentrating hormone (MCH) and melanin-concentrating hormone receptors (MCH-R) in mammalian skin physiology including pigmentation, inflammation and immune responses and skin cell proliferation. Much research is therefore still needed to define the roles of the hormone and its receptors in mammalian skin. This will be a crucial step to identifying pathogenic mechanisms that may involve the MCH/MCH-R system in the context of inflammatory and autoimmune skin diseases as well as skin cancers. The following review summarizes the studies which have been carried out to examine the expression and function of MCH and MCH-R in mammalian skin. Recent findings with regard to humoral immune responses to the MCH-R1 in patients with the skin depigmenting disease vitiligo are also discussed.