Maternal environment interacts with modifier genes to influence progression of nephrotic syndrome

Mutations in the NPHS2 gene, which encodes podocin, are responsible for some cases of sporadic and familial autosomal recessive steroid-resistant nephrotic syndrome. Inter- and intrafamilial variability in the progression of renal disease among patients bearing NPHS2 mutations suggests a potential role for modifier genes. Using a mouse model in which the podocin gene is constitutively inactivated, we sought to identify genetic determinants of the development and progression of renal disease as a result of the nephrotic syndrome. We report that the evolution of renal disease as a result of nephrotic syndrome in Nphs2-null mice depends on genetic background. Furthermore, the maternal environment significantly interacts with genetic determinants to modify survival and progression of renal disease. Quantitative trait locus mapping suggested that these genetic determinants may be encoded for by genes on the distal end of chromosome 3, which are linked to proteinuria, and on the distal end of chromosome 7, which are linked to a composite trait of urea, creatinine, and potassium. These loci demonstrate epistatic interactions with other chromosomal regions, highlighting the complex genetics of renal disease progression. In summary, constitutive inactivation of podocin models the complex interactions between maternal and genetically determined factors on the progression of renal disease as a result of nephrotic syndrome in mice.     

In vivo generation of neural tumors from neoplastic pluripotent stem cells models early human pediatric brain tumor formation

Recent studies have identified gene signatures in malignant tumors that are associated with human embryonic stem cells, suggesting a molecular relationship between aggressive cancers and pluripotency. Here, we characterize neural precursors (NPs) derived from transformed human embryonic stem cells (N-t-hESCs) that exhibit neoplastic features of human brain tumors. NPs derived from t-hESCs have enhanced cell proliferation and an inability to mature toward the astrocytic lineage, compared with progeny derived from normal human embryonic stem cells (N-hESCs) independent of adherent or neurosphere outgrowth. Intracranial transplantation of NPs derived from N-t-hESCs and N-hESCs into NOD SCID mice revealed development of neuroectoderm tumors exclusively from the N-t-hESCs NPs and not from normal N-hESCs. These tumors infiltrated the ventricles and the cerebellum of recipient mice and displayed morphological, phenotypic, and molecular features associated with classic medulloblastoma including retention of a pluripotent signature. Importantly, N-t-hESCs did not exhibit cytogenetic changes associated with medulloblastoma, suggesting that aberrant cellular and molecular properties precede the acquisition of karyotypic changes thus underscoring the value of this model system of human medulloblastoma. Our study demonstrates that NPs from a starting population of neoplastic human pluripotent parent cells possess brain tumor-initiating cell capacity, thereby providing a model system to investigate initiation and progression of primitive human neural cancers that are difficult to assess using somatic sources.     

Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line

Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.     

A medical-pharmaceutical partnership model as a contributor to the success in conditioning regimen for allogenic hematopoietic stem cell transplantation in adults: a cross-reflection on our organizations

Allogeneic hematopoietic stem-cell transplant (allo-SCT) remains the only cure for many hematological malignancies and some benign and congenital diseases. Busulfan, proposed in its injectable form, has quickly become a mainstay of pharmacological and myeloablative (or non-myeloablative) conditioning. This is following the outbreak in 2010 of a multicenter international clinical phase II trial, we tested the robustness and reliability of our organization in a complex model of organization and multifactorial partnership. In this type "BuCy2" protocol based on a classical treatment duration of 4 consecutive days, the administration of IV busulfan is given in one single daily infusion instead of the conventional 16 infusions, while keeping the same total dose. Under these conditions, the treatment is totally secured using a therapeutic drug monitoring of busulfan, applied in real-time. The process is technically complex and requires the very close cooperation of the teams involved. A strength, weakness, opportunity and threat (SWOT) analysis has been constructed; it fully supports continuous quality improvement to the triple benefit of the nursing chain, the patients and their environment. Several critical points were identified and corrected. The experiment strongly contributes to the safety and security of the medication circuit at the hospital and, improves the performance of allo-SCT. It also contributes to the protection of all actors in the health field and their working environment via a well-functioning quality management system.     

Syk-dependent mTOR activation in follicular lymphoma cells

The mammalian target of rapamycin (mTOR) is emerging as a promising target for antitumor therapy. However, the mechanism that contributes to its regulation in B lymphomas remains unknown. This study shows that in follicular lymphoma (FL) cells, mTOR is active because the cells displayed rapamycin-sensitive phosphorylation of p70S6 kinase and 4E-BP1. Moreover, immunohistochemistry applied on lymph node tissue sections obtained from patients with FL revealed that, in most cases, p70S6 kinase was highly phosphorylated compared to normal tonsillar tissue. In FL cells, mTOR was under control of both phospholipase D (PLD) and phosphatidylinositol 3-kinase (PI3K). Moreover, we demonstrated that Syk plays a central role in mTOR activation because we found that both expression and activity are elevated compared to normal or chronic lymphocytic leukemia B cells. We also provide evidence that Syk operates through PLD- and PI3K-independent pathways. Finally, Syk inhibition by piceatannol or by siRNA plasmids resulted in a potent inhibition of mTOR activity in FL cells, as well as in mantle cell lymphoma, Burkitt lymphoma, and diffuse large B-cell lymphoma. These findings suggest that the Syk-mTOR pathway has a critical function in FL survival, and therefore, that Syk could be a promising new target for B-lymphoma therapy.     

Dissecting and modeling photic and melanopsin effects to predict sleep disturbances induced by irregular light exposure in mice

Artificial lighting, day-length changes, shift work, and transmeridian travel all lead to sleep–wake disturbances. The nychthemeral sleep–wake cycle (SWc) is known to be controlled by output from the central circadian clock in the suprachiasmatic nuclei (SCN), which is entrained to the light–dark cycle. Additionally, via intrinsically photosensitive retinal ganglion cells containing the photopigment melanopsin (Opn4), short-term light–dark alternations exert direct and acute influences on sleep and waking. However, the extent to which longer exposures typically experienced across the 24-h day exert such an effect has never been clarified or quantified, as disentangling sustained direct light effects (SDLE) from circadian effects is difficult. Recording sleep in mice lacking a circadian pacemaker, either through transgenesis (Syt10^cre/creBmal1^fl/-) or SCN lesioning and/or melanopsin-based phototransduction (Opn4^−/−), we uncovered, contrary to prevailing assumptions, that the contribution of SDLE is as important as circadian-driven input in determining SWc amplitude. Specifically, SDLE were primarily mediated (>80%) through melanopsin, of which half were then relayed through the SCN, revealing an ancillary purpose for this structure, independent of its clock function in organizing SWc. Based on these findings, we designed a model to estimate the effect of atypical light–dark cycles on SWc. This model predicted SWc amplitude in mice exposed to simulated transequatorial or transmeridian paradigms. Taken together, we demonstrate this SDLE is a crucial mechanism influencing behavior on par with the circadian system. In a broader context, these findings mandate considering SDLE, in addition to circadian drive, for coping with health consequences of atypical light exposure in our society.

The nearly ubiquitous expression of circadian rhythmicity across species suggests that synchronizing physiology and behavior to Earth’s light–dark (LD) cycle is critical for survival and essential for optimal functioning and health. With increasing environmental light pollution and the introduction of new technologies such as light-emitting diodes and connected devices, human photic behavior is increasingly uncoupled from the natural LD cycle. The resulting perturbations in sleep–wake architecture have led to the increased prevalence of circadian disorders, insomnia, daytime somnolence, mood alteration, and poorer cognitive performance (1–5), stressing the need for a greater and more mechanistic understanding of the photic regulation of sleep and behavior.Light entrains the circadian pacemaker located in the suprachiasmatic nuclei (SCN), whose output signal generates an endogenous circadian sleep–wake rhythm aligned to the environmental LD cycle (6). However, light also exerts direct acute effects on sleep and waking, independent of the circadian system (7, 8). In nocturnal animals, such as most laboratory rodents, darkness administered for short periods (e.g., 1 h) acutely induces waking behavior, while the same length of light exposure promotes sleep (7). In circadian biology, these light- and/or dark-dependent changes were referred to as “masking,” as they can conceal the sleep–wake rhythm generated by the clock output signal. Therefore, this direct and noncircadian photic regulation was primarily considered through its indirect consequences on circadian function rather than a process actively imposing itself on the expression of sleep and waking in a direct and important manner (9). This highlights the need for a better understanding of the significance of the direct photic regulation of sleep and waking in comparison to clock-driven influence.The nonvisual effects of light are known to be mediated through different photoreceptive systems and neuronal pathways. Previous research led to the discovery of a novel retinal photoreceptor, melanopsin (Opn4), which is crucial for irradiance detection, maximally sensitive to blue light (480 nm), and expressed in a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) that convey nonimage-forming light information to the brain (10–12). These melanopsin ipRGCs project to various brain targets, which are thought to mediate light influence on physiology and behavior (13–15). Clock synchronization and circadian rhythm entrainment is mediated by projections to the SCN (16–18), whereas innervation of the sleep-promoting neurons of the ventrolateral preoptic area (VLPO) (19, 20) or the subparaventricular zone are thought to mediate light''s direct effects on sleep, yet it remains to be clarified whether the SCN might also participate in mediating those direct photic effects. Moreover, the loss of melanopsin does not abolish these responses, demonstrating that other photoreceptors involved in vision, the rods and cones, also play a role (12). Furthermore, the loss of ipRGCs results in dramatic loss of nonvisual effects, indicating that these cells receive inputs from rods and cones and are also the principal conduit for nonvisual light input from these photoreceptors (12, 18, 21–23). However, the degree of overlap between these two systems for regulating nonvisual responses is not yet certain.In recent years, the development of transgenic mouse models targeting these phototransduction pathways has revealed the pronounced effects of light and dark pulses of short duration on sleep and waking (11, 12, 24). Moreover, we previously showed that this acute and direct photic influence can be observed at all times of day (12). These observations raise the question as to whether longer periods of light and dark exposure, such as those of the 24-h LD cycle, exert sustained direct noncircadian effects, that is, continuous acute effects that could be observed over longer periods of time. One could postulate that the complete absence of light (constant darkness [DD] experiments) would allow for the extrapolation of the influence of sustained direct photic effects; however, this would not consider the possibility that the absence of light would lead to circadian disruption. Thus, under standard LD cycles, the contribution of direct noncircadian photic regulation in shaping the sleep–wake cycle has to date never been properly quantified due to the difficulty in disentangling it from the circadian effects.In the current study, we sought to address three related issues: 1) the quantification of the respective contribution of circadian effects (CE) and sustained direct light effects (SDLE) in shaping the 24-h sleep–wake cycle; 2) the identification of the pathways underlying those effects (i.e., the respective role of the photoreceptive systems), melanopsin versus rods and cones, and the respective role of the SCN versus non-SCN brain relays as conduits for this direct photic regulation of the sleep–wake cycle; and 3) whether quantifying the respective contributions of CE and SDLE allows for the prediction of the nychthemeral sleep–wake distribution under unnatural LD cycles, such as those experienced during jet lag. Recording sleep in mice lacking a functional central clock either through SCN lesioning or Bmal1 tissue-specific clock gene deletion and/or melanopsin-based phototransduction, we uncovered that CE and SDLE contribute in equal proportion to shaping the nychthemeral cycle. Furthermore, analyzing sleep in melanopsin-deficient mice with or without disabled SCN revealed that SDLE are primarily (>80%) mediated through melanopsin-based phototransduction, half of which passes via the SCN, implying a noncircadian function for the brain structure comprising the master circadian clock. To further validate our findings, these data were integrated into a model that accurately predicted 24-h sleep–wake distribution in mice exposed to a simulated “jet lag” or transequatorial travel paradigm.     

Differential cellular responses induced by dorsomorphin and LDN‐193189 in chemotherapy‐sensitive and chemotherapy‐resistant human epithelial ovarian cancer cells

Inherent or acquired drug resistance is a major contributor to epithelial ovarian cancer (EOC) mortality. Novel drugs or drug combinations that produce EOC cell death or resensitize drug resistant cells to standard chemotherapy may improve patient treatment. After conducting drug tolerability studies for the multikinase inhibitors dorsomorphin (DM) and it is structural analogue LDN‐193189 (LDN), these drugs were tested in a mouse intraperitoneal xenograft model of EOC. DM significantly increased survival, whereas LDN showed a trend toward increased survival. In vitro experiments using cisplatin (CP)‐resistant EOC cell lines, A2780‐cp or SKOV3, we determined that pretreatment or cotreatment with DM or LDN resensitized cells to the killing effect of CP or carboplatin (CB). DM was capable of blocking EOC cell cycle and migration, whereas LDN produced a less pronounced effect on cell cycle and no effect on migration. Subsequent analyses using primary human EOC cell samples or additional established EOC cells lines showed that DM or LDN induced a dose‐dependent autophagic or cell death response, respectively. DM induced a characteristic morphological change with the appearance of numerous LC3B‐containing acidic vacuoles and an increase in LC3BII levels. This was coincident with a decrease in cell growth and the altered cell cycle consistent with DM‐induced cytostasis. By contrast, LDN produced a caspase 3‐independent, reactive oxygen species‐dependent cell death. Overall, DM and LDN possess drug characteristics suitable for adjuvant agents used to treat chemotherapy‐sensitive and ‐resistant EOC.