HIV-1 reverse transcriptase connection domain mutations: dynamics of emergence and implications for success of combination antiretroviral therapy.

BACKGROUND: Factors promoting the emergence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) connection domain mutations and their effect on antiretroviral therapy (ART) are still largely undetermined. We investigated this matter by analyzing genotypic resistance tests covering 400 amino acid positions in the RT of HIV-1 subtype B viruses and corresponding treatment histories and laboratory measurements. METHODS: The emergence of connection domain mutations was studied in 334 patients receiving monotherapy or dual therapy with thymidine analogues at the time of the genotypic resistance test. Response to subsequent combination ART (cART) was analyzed using Cox regression for 291 patients receiving unboosted protease inhibitors. Response was defined by ever reaching an HIV RNA level <50 copies/mL during the first cART. RESULTS: The connection domain mutations N348I, R356K, R358K, A360V, and A371V were more frequently observed in ART-exposed than ART-naive patients, of which only N348I and A360V were nonpolymorphic (with a prevalence of <1.5% in untreated patients). N348I correlated with M184V and predominantly occurred in patients receiving lamivudine and zidovudine concomitantly. A360V was not associated with specific drug combinations and was found to emerge later than M184V or thymidine analogue mutations. Nonpolymorphic connection domain mutations were rarely detected in the absence of established drug resistance mutations in ART-exposed individuals (prevalence, <1%). None of the 5 connection domain mutations associated with treatment showed a statistically significant effect on response to cART. CONCLUSIONS: Despite their frequent emergence, connection domain mutations did not show large detrimental effects on response to cART. Currently, routine implementation of connection domain sequencing seems unnecessary for developed health care settings.

Actin filament dynamics impacts keratinocyte stem cell maintenance.

Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1.

Niche dynamics in space and time.

Niche conservatism, the tendency of a species niche to remain unchanged over time, is often assumed when discussing, explaining or predicting biogeographical patterns. Unfortunately, there has been no basis for predicting niche dynamics over relevant timescales, from tens to a few hundreds of years. The recent application of species distribution models (SDMs) and phylogenetic methods to analysis of niche characteristics has provided insight to niche dynamics. Niche shifts and conservatism have both occurred within the last 100 years, with recent speciation events, and deep within clades of species. There is increasing evidence that coordinated application of these methods can help to identify species which likely fulfill one key assumption in the predictive application of SDMs: an unchanging niche. This will improve confidence in SDM-based predictions of the impacts of climate change and species invasions on species distributions and biodiversity.

The ALK-F1174L activating mutation is tumorigenic in MONC-1 neural crest stem cells in an orthotopic murine model of neuroblastoma

Background: Activating mutations of the anaplastic lymphoma receptor tyrosine kinase gene (ALK) were identified in both somatic and familial neuroblastoma. The most common somatic mutation, F1174L, is associated with NMYC amplification and displayed an efficient transforming activity in vivo. In addition, both AKL-F1174L and NMYC were shown cooperate in neuroblastoma tumorigenesis in animal models. To analyse the role of ALK mutations in the oncogenesis of neuroblastoma, ALK wt and various ALK mutants were transduced in murine neural crest stem cells (MONC1). Methods: ALK-wt, and F1174L, and R1275Q mutants were stably expressed by retroviral infection using the pMIGR1 vector in the murine neural crest stem cell line MONC-1, previously immortalised with v-myc, and further implanted subcutaneously or orthotopically in nude mice. Results: Both MONC1-ALK-F1174L and -R1275Q cells displayed a rapid tumour forming capacity upon subcutaneous injection in nude mice compared to control MONC1-MIGR or MONC1 cells. Interestingly, the transforming capacity of the F1174L mutant was much more potent compared to that of R1275Q mutant in murine neural crest stem cells, while ALK-wt was not tumorigenic. In addition, mice implanted orthotopically in the left adrenal gland with MONC1-ALK-F1174L cells developed highly aggressive tumours in 100% of mice within three weeks, while MONC1-Migr or MONC1 derived tumours displayed a longer latency and a reduced tumour take. Conclusions: The activating ALK-F1174L mutant is highly tumorigenic in neural crest stem cells. Nevertheless, we cannot exclude a functional implication of the v-myc oncogene used for MONC1 cells immortalisation. Indeed, the control MONC1-Migr and MONC1 cells were also able to derive subcutaneous and orthotopic tumours, although with considerable reduced efficiency. Further investigations using neural crest stem cell lacking exogenous myc expression are currently on way to assess the exclusive role of ALK mutations in NB oncogenesis.

Effect of roscovitine on intracellular calcium dynamics: differential enantioselective responses.

Cyclin-dependent kinases (CDKs) inhibitors have emerged as interesting therapeutic candidates. Of these, (S)-roscovitine has been proposed as potential neuroprotective molecule for stroke while (R)-roscovitine is currently entering phase II clinical trials against cancers and phase I clinical tests against glomerulonephritis. In addition, (R)-roscovitine has been suggested as potential antihypertensive and anti-inflammatory drug. Dysfunction of intracellular calcium balance is a common denominator of these diseases, and the two roscovitine enantiomers (S and R) are known to modulate calcium voltage channel activity differentially. Here, we provide a detailed description of short- and long-term responses of roscovitine on intracellular calcium handling in renal epithelial cells. Short-term exposure to (S)-roscovitine induced a cytosolic calcium peak, which was abolished after stores depletion with cyclopiazonic acid (CPA). Instead, (R)-roscovitine caused a calcium peak followed by a small calcium plateau. Cytosolic calcium response was prevented after stores depletion. Bafilomycin, a selective vacuolar H(+)-ATPase inhibitor, abolished the small calcium plateau. Long-term exposure to (R)-roscovitine significantly reduced the basal calcium level compared to control and (S)-roscovitine treated cells. However, both enantiomers increased calcium accumulation in the endoplasmic reticulum (ER). Consistently, cells treated with (R)-roscovitine showed a significant increase in SERCA activity, whereas (S)-roscovitine incubation resulted in a reduced PMCA expression. We also found a tonic decreased ability to release calcium from the ER, likely via IP3 signaling, under treatment with (S)- or (R)-roscovitine. Together our data revealed that (S)-roscovitine and (R)-roscovitine exert distinct enantiospecific effects on intracellular calcium signaling in renal epithelial cells. This distinct pharmacological profile can be relevant for roscovitine clinical use.

NO rebinding to myoglobin: a reactive molecular dynamics study.

The rebinding of NO to myoglobin after photolysis is studied using the 'reactive molecular dynamics' method. In this approach the energy of the system is evaluated on two potential energy surfaces that include the heme-ligand interactions which change between liganded and unliganded myoglobin. This makes it possible to take into account in a simple way, the high dimensionality of the transition seam connecting the reactant and product states. The dynamics of the dissociated NO molecules are examined, and the geometrical and energetic properties of the transition seam are studied. Analysis of the frequency of recrossing shows that the height of the effective rebinding barrier is dependent on the time after photodissociation. This effect is due mainly to protein relaxation and may contribute to the experimentally observed non-exponential rebinding rate of NO, as has been suggested previously.

Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13.

Genomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICEclc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICEclc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICEclc can target a number of different tRNA(Gly) genes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICEclc based on the attR sequence, and we could show that ICEclc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICEclc is excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, including Bordetella petri, Pseudomonas aeruginosa and Burkholderia.

Lifelong dynamics of human CD4+CD25+ regulatory T cells: insights from in vivo data and mathematical modeling.

Despite their limited proliferation capacity, regulatory T cells (T(regs)) constitute a population maintained over the entire lifetime of a human organism. The means by which T(regs) sustain a stable pool in vivo are controversial. Using a mathematical model, we address this issue by evaluating several biological scenarios of the origins and the proliferation capacity of two subsets of T(regs): precursor CD4(+)CD25(+)CD45RO(-) and mature CD4(+)CD25(+)CD45RO(+) cells. The lifelong dynamics of T(regs) are described by a set of ordinary differential equations, driven by a stochastic process representing the major immune reactions involving these cells. The model dynamics are validated using data from human donors of different ages. Analysis of the data led to the identification of two properties of the dynamics: (1) the equilibrium in the CD4(+)CD25(+)FoxP3(+)T(regs) population is maintained over both precursor and mature T(regs) pools together, and (2) the ratio between precursor and mature T(regs) is inverted in the early years of adulthood. Then, using the model, we identified three biologically relevant scenarios that have the above properties: (1) the unique source of mature T(regs) is the antigen-driven differentiation of precursors that acquire the mature profile in the periphery and the proliferation of T(regs) is essential for the development and the maintenance of the pool; there exist other sources of mature T(regs), such as (2) a homeostatic density-dependent regulation or (3) thymus- or effector-derived T(regs), and in both cases, antigen-induced proliferation is not necessary for the development of a stable pool of T(regs). This is the first time that a mathematical model built to describe the in vivo dynamics of regulatory T cells is validated using human data. The application of this model provides an invaluable tool in estimating the amount of regulatory T cells as a function of time in the blood of patients that received a solid organ transplant or are suffering from an autoimmune disease.

Dynamics of IS Adaptation in Multinational Corporations: A New Theoretical Lens

The specificities of multinational corporations (MNCs) have to date not been a focus area of IS research. Extant literature mostly proposes IS configurations for specific types of MNCs, following a static and prescriptive approach. Our research seeks to explain the dynamics of global IS design. It suggests a new theoretical lens for studying global IS design by applying the structural adjustment paradigm from organizational change theories. Relying on archetype theory, we conduct a longitudinal case study to theorize the dynamics of IS adaptation. We find that global IS design emerges as an organizational adaptation process to balance interpretative schemes (i.e. the organization's values and beliefs) and structural arrangements (i.e. strategic, organizational, and IS configurations). The resulting insights can be used as a basis to further explore alternative global IS designs and movements between them.