Extending reference assembly models

The human genome reference assembly is crucial for aligning and analyzing sequence data, and for genome annotation, among other roles. However, the models and analysis assumptions that underlie the current assembly need revising to fully represent human sequence diversity. Improved analysis tools and updated data reporting formats are also required.     

Anti-tumor and anti-leishmanial evaluations of 1,3,4-oxadiazine, pyran derivatives derived from cross-coupling reactions of β-bromo-6H-1,3,4-oxadiazine derivatives

Cyanoacetylhydrazine reacted with the ω-bromoacetophenones 2a,b to give hydrazide-hydrazone derivatives 3a,b. The latter products were cyclized to the 1,3,4-oxadiazine derivatives 4a,b. Bromination of the latter products gave the 6-bromo-6H-1,3,4-oxadiazine derivatives 5a,b which underwent a series of cross-coupling reactions. The antitumor evaluation of the newly synthesized products against the three cancer cells namely breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and CNS cancer (SF-268) showed that some of them have high inhibitory effect towards three cell lines which is higher than the standard. Moreover, the anti-leishmanial activity of the newly synthesized product was tested on Leishmania donovani amastigotes showed that some compounds have high activity.     

Recombination centres and the orchestration of V(D)J recombination

The initiation of V(D)J recombination by the recombination activating gene 1 (RAG1) and RAG2 proteins is carefully orchestrated to ensure that antigen receptor gene assembly occurs in the appropriate cell lineage and in the proper developmental order. Here we review recent advances in our understanding of how DNA binding and cleavage by the RAG proteins are regulated by the chromatin structure and architecture of antigen receptor genes. These advances suggest novel mechanisms for both the targeting and the mistargeting of V(D)J recombination, and have implications for how these events contribute to genome instability and lymphoid malignancy.     

Targeted cancer therapy: What if the driver is just a messenger?

“Shoot the driver” is the paradigm of targeted cancer therapy. However, resistance to targeted inhibitors of signaling pathways is a major problem. In part, the redundancy of signaling networks can bypass targeted inhibitors and thereby reduce their biological effect. In this case, the driver turns out to be one of several potential messengers and is easily replaced. Cocktails of multiple targeted inhibitors are an obvious solution. This is limited, however, by the lack of potent inhibitors and may also produce increased toxicity. Therefore, we explored the direct blockade of a key biological activity downstream from multiple converging oncogenic signals. Specifically, several oncogenic signaling pathways, including AKT, MAPK and PIM kinase signals, converge on the activation of cap-dependent translation. In cancer cells, aberrant activation of cap-dependent translation favors the increased expression of short-lived oncoproteins like c-MYC, MCL1, CYCLIND1 and the PIM kinases. Intriguingly, cancer cells are especially sensitive to even temporary reductions in these proteins. We will discuss our findings concerning translational inhibitor therapy in cancer.Key words: targeted therapy, cancer, lymphoma, translation, eIF4ETargeted cancer therapies are designed to block selected pathways or molecules that are required for tumor cell survival. The most successful examples are inhibitors of the BCR-ABL fusion protein that drive chronic myeloid leukemia (CML). By shutting down the activity of a single molecule driving the growth of CML cells, imatinib and its successors dasatinib and nilotinib can produce complete and sustained remissions as single agents (reviewed in ref. ¹). However, it has proven difficult to translate this success to other cancers. In metastatic melanoma, for example, the BRAF inhibitor vemurafenib produces high response rates in patients whose tumors bear the BRAF-V600E activating mutation (reviewed in ref. ²). Excitement generated by these results is well-deserved, as they have opened a new treatment paradigm in a disease with few available options and a grim prognosis. Unfortunately, however, resistance to the drug generally is seen within a few months. Median progression-free survival resulting from vemurafenib for treatment-naïve metastatic melanoma patients with V600E was about six months,³ compared with imatinib in CML, which kept 93% of patients progression-free at five years.⁴ The success of TKIs in CML is still the exception and not the rule in targeted cancer therapeutics.Resistance to targeted inhibitors is an emerging problem with multiple causes and potential solutions. While mechanisms of resistance to BRAF inhibition in melanoma remain to be elucidated, they are well-described for some targets in other cancers. Drugs against the epidermal growth factor receptor (EGFR) kinase, for example, are bypassed both by activation of downstream mediators, most prominently KRAS, and by signaling through parallel pathways like the MET oncogene (reviewed in ref. ⁵). Resistance mechanisms are varied and complex in some ways, but most boil down to the same idea: evolution has provided multiple routes to the crucial endpoints that allow sustained growth and proliferation of cancer cells. Cocktails of multiple inhibitors have been proposed to prevent or thwart resistance (reviewed in ref. ⁶), and this approach appears highly promising based on some recent preclinical studies. In prostate cancer, for example, combined blockade of androgen receptor signaling and the PI3K/AKT/mTOR pathway showed potent synergy in model systems.⁷ Unfortunately, similar approaches against many other cancers are currently limited by the availability of potent, selective inhibitors and a need to identity which combinations will be effective. Moreover, combined toxicities from simultaneous use of multiple inhibitors will pose limits on the number and intensity of drugs that can be used.An alternative and potentially complementary approach is to directly target the downstream biological processes that are activated by signaling pathways and that cancer cells rely on. Experiences with inhibitors of EGFR, BRAF, and other signaling molecules suggest that most tumors can reliably activate parallel or downstream messengers to thwart efficacy. So inhibiting a signaling intermediate (i.e., a messenger) allows resistance if the biological effects can be achieved via an alternate route. Ultimately, tumor cells don''t depend on the messenger, often a kinase, and, instead, require a downstream biological function. This opens the possibility that targeting the critical effect directly may be an effective cancer therapy and could overcome the problem of redundant messengers.Several inherent properties of signaling pathways are relevant to targeted therapies, their side effects and mechanisms of resistance.⁸ For example, signaling pathways frequently converge on key activities. As explained above, this redundancy can produce resistance to targeted agents. Often redundant pathways are induced via feedback mechanisms, which provide robust signals and can also bypass selective inhibitors. Signals also diverge, however, and seemingly parallel pathways therefore also produce pleiotropic and non-overlapping effects. It is possible not all activities are required by cancer cells. Hence, an upstream block may produce toxicities, in part, by blocking activities that are not strictly required by tumor cells as much as by normal tissues. Finally, unlike metabolic pathways, where a limiting substrate is passed down, signaling cascades amplify signals toward a key activity, and the initial signal or message is both energetically “cheap” and infinitely recurrent. Accordingly, it is conceivable that direct block of the downstream effects provide an alternative or complementary approach to targeting upstream signaling molecules.Multiple oncogenic signals, including the PI3K, MAPK/ERK and PIM kinase pathways converge on the activation of cap-dependent translation, the process by which most capped mRNAs are translated into proteins (reviewed in ref. ⁹). Signaling pathways control the availability of the cap-binding protein eIF4E that is the limiting component of the multimeric translation complex eIF4F, which also includes scaffolds (eIF4G) and RNA helicase activities (eIF4A).^10–12 The complex ultimately mediates loading of mRNAs onto ribosomes. Availability of the eIF4E factor is especially important for mRNAs with long and structured 5′ UTRs. These include, in particular, short-lived cell cycle regulators and oncoproteins. Hence, regulation of eIF4E via upstream signals provides an immediate level of expression control that directly controls levels of proteins, including c-MYC, cyclin D1, BCL2, MCL1 and PIM1.^13–17 Cancer cells require continuous expression of these proteins. For example, even brief loss of MYC expression produces widespread cell death in several cancers but only produces reversible cell cycle arrest in normal tissues.¹⁸ Hence, the increased requirement for the continuous translation of oncoproteins in cancer cells may provide a therapeutic window for inhibitors of capdependent translation.We recently investigated the therapeutic potential of directly blocking capdependent translation in non-Hodgkin lymphoma (NHL).^15,16,19 Rapalogs, inhibitors of mTORC1, the upstream activator of cap-dependent translation, have been extensively studied in NHL in clinical trials (reviewed in ref. ²⁰). However, their activity has, overall, been modest, and our results implicate mTORC1-independent activation of translation by PIM kinases as one mechanism of rapalog resistance in NHLs.¹⁶ The PIM family kinases are active upon expression and do not require activating modifications. They have been known for some time to be able to promote phosphorylation of 4E-BP1 in a manner resistant to rapamycin.^21,22 We now report expression of PIM1 and/or PIM2 in more than 60% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) and more than 75% of mantle cell and small lymphocytic lymphomas. Our study found PIM expression (either PIM1 or PIM2) was associated with worse time to event and overall survival in FL, while another recent report points to PIM2 as a driver of aggressive disease in activated B-cell type DLBCL.²³ In addition, two recent studies of chromosomal translocations mediated by activation-induced deaminase (AID) identified PIM1 as a frequent target.^24,25 In sum, expression of PIM kinases is common in NHLs, may be associated with a more aggressive clinical course and exemplifies how the redundancy of messaging molecules can bypass the clinical activity of selective signaling inhibitors.In experimental systems, we found that direct blockade of cap-dependent translation was highly effective against lymphomas with redundant PI3K/AKT and PIM signals.¹⁶ Briefly, using both a constitutively active mutant 4E-BP1 allele that blocks eIF4E activity and a small molecule inhibitor of the eIF4A helicase, silvestrol, we were able to completely restore rapalog sensitivity in lymphomas engineered to express PIM2 kinase activity. Mechanistically, we found that silvestrol dramatically reduced the translation of critical oncoproteins, including c-MYC, cyclin D1 and MCL1. Interestingly, silvestrol also blocked the translation of both PIM kinases themselves. Moreover, consistent with prior reports, silvestrol treatment at an effective dose was well-tolerated in animals, and we observed no frank toxicity.¹⁹ Hence, blocking cap-dependent translation disrupts upstream signaling molecules, the PIM kinases and also key oncoproteins commonly considered “undruggable” oncoproteins, including c-MYC.Silvestrol worked dramatically better than inhibition of the upstream kinases. Briefly, we tested the SuperGen Inc. PIM kinase inhibitors SGI-1776 and SGI-1773 side by side with silvestrol in a panel of these PIM-expressing human NHL cell lines. Notably, SGI-1776 is the only PIM inhibitor that has entered clinical trials, although these had to be discontinued due to cardiac toxicity of the compound (SuperGen press release, 2010). In any case, silvestrol showed in vitro potency at IC₅₀ of less than 10 nM in all cases, and the PIM kinase inhibitors were 100 to 1,000 times less active. These results highlight some problems associated with the “inhibitor cocktail” approach and indicate a complementary strategy that includes direct blockade of a key biological activity, in this instance, cap-dependent translation of oncoproteins.Silvestrol is not the only means to block cap-dependent translation, and others are reviewed in reference ⁹. These include antisense oligonucleotides against eIF4E and peptide inhibitors of eIF4F complex formation, though neither has entered clinical trials. Silvestrol is the most well-studied of several compounds that emerged from library screens for ability to disrupt the function of the eIF4F subunit eIF4A, an RNA helicase required for its ability to promote mRNA translation. A plant-derived flavagline, silvestrol has shown activity against a variety of tumor types and can be given to mice at high enough concentrations for antitumor activity without major toxicity. The drug shows activity as a single agent against human breast and prostate cancer cell lines in xenograft experiments in nude mice.²⁶ This produced mild transient impairment of hepatic synthetic function but no toxicities producing morbidity or mortality. In genetically defined murine tumor models, silvestrol showed potent synergy with chemotherapy when used against tumors bearing translational activation due to loss of Pten or overexpression of eIf4e.¹⁹ Originally isolated from Aglaia silvestris, silvestrol has a complex structure that has proved difficult to chemically synthesize in quantity. For this reason, the parent compound is not an ideal clinical drug candidate. Efforts are underway by Drs. Pelletier (McGill) and Porco (Boston University) to develop analogs with more efficient synthesis profiles and that retain its biochemical properties. In sum, cap-dependent translation is a promising drug target alternate to mTORC1 and upstream kinase inhibitors.     

Quantification of optic disc edema during exposure to high altitude shows no correlation to acute mountain sickness

Background

The study aimed to quantify changes of the optic nerve head (ONH) during exposure to high altitude and to assess a correlation with acute mountain sickness (AMS). This work is related to the Tuebingen High Altitude Ophthalmology (THAO) study.

Methodology/Principal Findings

A confocal scanning laser ophthalmoscope (cSLO, Heidelberg Retina Tomograph, HRT3®) was used to quantify changes at the ONH in 18 healthy participants before, during and after rapid ascent to high altitude (4559 m). Slitlamp biomicroscopy was used for clinical optic disc evaluation; AMS was assessed with Lake Louise (LL) and AMS-cerebral (AMS-c) scores; oxygen saturation (SpO₂) and heart rate (HR) were monitored. These parameters were used to correlate with changes at the ONH. After the first night spent at high altitude, incidence of AMS was 55% and presence of clinical optic disc edema (ODE) 79%. Key stereometric parameters of the HRT3® used to describe ODE (mean retinal nerve fiber layer [RNFL] thickness, RNFL cross sectional area, optic disc rim volume and maximum contour elevation) changed significantly at high altitude compared to baseline (p<0.05) and were consistent with clinically described ODE. All changes were reversible in all participants after descent. There was no significant correlation between parameters of ODE and AMS, SpO₂ or HR.

Conclusions/Significance

Exposure to high altitude leads to reversible ODE in the majority of healthy subjects. However, these changes did not correlate with AMS or basic physiologic parameters such as SpO₂ and HR. For the first time, a quantitative approach has been used to assess these changes during acute, non-acclimatized high altitude exposure. In conclusion, ODE presents a reaction of the body to high altitude exposure unrelated to AMS.     

Fourier shell correlation threshold criteria

The resolution value claimed for an electron microscopical three-dimensional reconstruction indicates the overall quality of the experiment. The Fourier shell correlation (FSC) criterion has now become the standard quality measure. However, what has continued to be controversial is the issue of the FSC threshold level at which one defines the reproducible resolution. Here, we discuss the theoretical behaviour of the FSC in conjunction with the various factors which influence it: the number of "voxels" in a given Fourier shell, the symmetry of the structure, and the size of the structure within the reconstruction volume. Both the theoretical considerations and our model experiments show that fixed-valued FSC threshold (like "0.5") may never be used in a reproducible criterion. Fixed threshold values are-as we show here-simply the result of incorrect assumptions in the basic statistics. Two families of FSC threshold curves are discussed: the sigma-factor curves and the new family of bit-based information threshold curves. Whereas sigma-factor curves indicate the resolution level at which one has collected information significantly above the noise level, the information curves indicate the resolution level at which enough information has been collected for interpretation.     

Ecological implications of the emergence of non-toxic subcultures from toxic Microcystis strains

Two toxic, microcystin-producing, Microcystis sp. strains KLL MG-K and KLL MB-K were isolated as single colonies on agar plates from Lake Kinneret, Israel. Two non-toxic subcultures, MG-J and MB-J spontaneously succeeded the toxic ones under laboratory conditions. Southern analyses showed that MG-J and MB-J are lacking at least 34 kb of the mcy region, encoding the microcystin synthetase. Analyses of the 16S rRNA genes, the intergenic spacer region between cpcB and cpcA and the patterns of the polymerase chain reaction products of randomly amplified polymorphic DNA and highly iterated palindrome, and presence of mobile DNA elements did not allow unequivocal distinction between toxic and non-toxic subcultures. Laboratory and field experiments indicated an advantage of the toxic strain over its non-toxic successor. When grown separated by a membrane, which allowed passage of the media but not the cells, MG-K severely inhibited the growth of MG-J. Furthermore, when MG strains were placed in dialysis bags in Lake Kinneret during the season in which Microcystis is often observed, cells of MG-J lysed, whereas MG-K survived. Mechanisms whereby the non-toxic subcultures emerged and prevailed over the corresponding toxic ones under laboratory conditions, as well as a possible role of microcystin under natural conditions, are discussed.