Targeting MYC-driven replication stress in medulloblastoma with AZD1775 and gemcitabine

Journal of Neuro-Oncology - MYC-driven medulloblastomas are highly aggressive childhood tumors with dismal outcomes and a lack of new treatment paradigms. We identified that targeting replication...     

Making the decision to donate eyes: Perspectives from the families of the deceased in Madurai,India

Purpose:To identify factors affecting family members'' decision whether to donate eye organs.Methods:A community-based case-control study based on in-home interviews with families of deceased individuals who had or had not donated eye organs, in Madurai district, Tamil Nadu, India. Data collected were knowledge and awareness of eye donations, whether the deceased individual had expressed or pledged willingness to donate, and family members'' attitudes and willingness to donate their own eye organs.Results:Seventy-six families of donors and 256 families of non-donors completed the survey. Multivariable analysis showed that the following variables were significantly associated with a donation: age, whether the deceased had registered for eye donation, pre-expressed willingness of deceased to donate, whether family members personally know beneficiaries of eye donations, and higher score on a scale evaluating knowledge and awareness about eye donation. The majority of donors'' families (71%) had been encouraged by someone to donate. Among non-donor families, a substantially larger fraction (52.8%) indicated they would have donated had someone reminded or encouraged them to do so, in comparison with those who indicated lack of awareness or knowledge (14.5%).Conclusion:Community programs are likely to be effective if they encourage individuals to pledge their eyes or express their willingness to donate their eyes to family members in advance of death; they increase public awareness of the value of eye donation. A friend, family member, neighbor or counselor approaching bereaved families and having a dialogue about eye donation would substantially increase the probability of a decision to donate.     

E1 of α-ketoglutarate dehydrogenase defends Mycobacterium tuberculosis against glutamate anaplerosis and nitroxidative stress

Enzymes of central carbon metabolism (CCM) in Mycobacterium tuberculosis (Mtb) make an important contribution to the pathogen’s virulence. Evidence is emerging that some of these enzymes are not simply playing the metabolic roles for which they are annotated, but can protect the pathogen via additional functions. Here, we found that deficiency of 2-hydroxy-3-oxoadipate synthase (HOAS), the E1 component of the α-ketoglutarate (α-KG) dehydrogenase complex (KDHC), did not lead to general metabolic perturbation or growth impairment of Mtb, but only to the specific inability to cope with glutamate anaplerosis and nitroxidative stress. In the former role, HOAS acts to prevent accumulation of aldehydes, including growth-inhibitory succinate semialdehyde (SSA). In the latter role, HOAS can participate in an alternative four-component peroxidase system, HOAS/dihydrolipoyl acetyl transferase (DlaT)/alkylhydroperoxide reductase colorless subunit gene (ahpC)-neighboring subunit (AhpD)/AhpC, using α-KG as a previously undescribed source of electrons for reductase action. Thus, instead of a canonical role in CCM, the E1 component of Mtb’s KDHC serves key roles in situational defense that contribute to its requirement for virulence in the host. We also show that pyruvate decarboxylase (AceE), the E1 component of pyruvate dehydrogenase (PDHC), can participate in AceE/DlaT/AhpD/AhpC, using pyruvate as a source of electrons for reductase action. Identification of these systems leads us to suggest that Mtb can recruit components of its CCM for reactive nitrogen defense using central carbon metabolites.The bacterium Mycobacterium tuberculosis (Mtb), which causes tuberculosis, has plagued humanity since antiquity (1), is estimated to infect one-third of the population today, and is the leading cause of death by a bacterium. This success as a pathogen reflects Mtb’s metabolic plasticity and resistance to host immunity (2, 3). Recent evidence suggests that certain enzymes of central carbon metabolism (CCM) can mediate both of these facets of Mtb’s adaptation to the host (4–8). Here, we demonstrate that Rv1248c, recently named 2-hydroxy-3-oxoadipate synthase (HOAS) (9), is one such enzyme.Rv1248c was first annotated as the thiamin diphosphate (ThDP)-dependent E1 component (SucA) of a canonical α-ketoglutarate (α-KG) dehydrogenase complex (KDHC) that produces succinyl CoA (SucCoA) via oxidative decarboxylation of α-KG with concomitant transfer of the resulting succinyl group to CoA (10). Classically, KDHC, composed of three enzymes, joins the oxidative and reductive half-cycles of the TCA cycle (SI Appendix, Fig. S1). The TCA cycle generates high-energy phosphate bonds and biosynthetic precursors of amino acids, nucleotides, and fatty acids (11). However, KDHC activity was not detected in Mtb lysates, and the gene product annotated as the lipoamide-bearing E2 component [Rv2215, dihydrolipoyl succinyl transferase (SucB)] functions as the E2 component dihydrolipoyl acetyl transferase (DlaT) of the pyruvate dehydrogenase complex (PDHC) (5, 12, 13). The Mtb genome does not encode another SucB (12), and KDHC activity could not be demonstrated with purified recombinant Rv1248c plus DlaT and E3 [lipoamide dehydrogenase (Lpd)] in a manner similar to cognate proteins from other actinomycetes (14). Rv1248c by itself produces succinate semialdehyde (SSA) from nonoxidative decarboxylation of α-KG in vitro, and Mtb’s succinate semialdehyde dehydrogenases (SSADHs) can generate succinate from SSA, completing a modified TCA cycle (13). Subsequently, activity-based metabolomic profiling revealed yet another function of Rv1248c that predominated over SSA production: decarboxylation of α-KG, followed by carboligation with glyoxylate to form 2-hydroxy-3-oxoadipate (HOA). Thus, Rv1248c was renamed HOAS (9). Decarboxylation of α-KG is the first step in all three reactions. The Mycobacterium smegmatis α-ketoglutarate decarboxylase (MsKGD) homolog was found to catalyze all three reactions in vitro, with augmentation of catalysis by acetyl CoA (AcCoA)-mediated allosteric modulation (15), whereas Mtb HOAS showed a kinetic preference for the HOAS pathway in vitro (16).HOAS activity is regulated in Mtb by glycogen accumulation regulator A (GarA), whose activity is controlled, in turn, by Ser/Thr kinases PknG and PknB (SI Appendix, Fig. S1B). GarA also regulates glutamate dehydrogenase (GDH) and glutamate synthase/glutamine oxoglutarate aminotransferase (17). Coregulation of these three enzymes by GarA calls attention to the contribution of HOAS to Mtb’s metabolism of glutamate. Glutamate serves as an anaplerotic substrate entering the TCA cycle as α-KG and is also a key intermediate in nitrogen assimilation and metabolism (18, 19).Despite extensive studies, the physiological function of HOAS in Mtb and its contribution to virulence remain unknown. Here, we brought genetics, metabolomics, enzymology, and mouse models of infection to bear on that question, using the hoas deletion mutant in Mtb and the deletion mutant complemented in three ways: with the WT allele, with an allele with a point mutation that abrogates catalysis, or with an allele with a point mutation that is insensitive to allosteric regulation by AcCoA. In standard culture conditions, Δhoas showed no defect in growth or changes in levels of CCM metabolites, arguing against Mtb’s reliance on the conventional function of KDHC. However, situational stresses revealed striking phenotypes in Δhoas, indicative of a defensive role of HOAS against products arising from metabolism of glutamate and against reactive nitrogen intermediates (RNIs). Mechanistic analysis of the latter phenotype revealed a previously undescribed route to antioxidant defense mediated by substrates and enzymes of CCM.     

AT-101 induces apoptosis in CLL B cells and overcomes stromal cell-mediated Mcl-1 induction and drug resistance

Resistance to apoptosis in CLL B cells is associated with overexpression of Bcl-2 family antiapoptotic proteins. Their expression is endogenous, but is also induced by signals from the microenvironment resulting in intrinsic and extrinsic drug resistance. Because AT-101 binds to the BH3 motif of all Bcl-2-family antiapoptotic proteins, we hypothesized that this molecule could overcome resistance. AT-101 treatment (20 microM for 24 hours) resulted in a median 72% apoptosis in CLL cells (patients; n = 32, P < .001). Stromal cells protected CLL B cells from spontaneous and fludarabine-induced apoptosis (P = .003) by increasing the Mcl-1 protein levels. However, AT-101 induced similar extent of down-regulation of Mcl-1 and apoptosis in CLL lymphocytes cultured in suspension or on stroma (P = .999). Stromal cells expressed undetectable levels of antiapoptotic but high levels of activated ERK and AKT proteins and had low or no apoptosis with AT-101. Collectively, these data demonstrate that AT-101 induces apoptosis in CLL B cells and overcomes microenvironment-mediated resistance while sparing normal stromal cells.     

Bias in estimation of transporter kinetic parameters from overexpression systems: Interplay of transporter expression level and substrate affinity.

The objective was to investigate the interplay between transporter expression levels and substrate affinity in controlling the influence of aqueous boundary layer (ABL) resistance on transporter kinetics in an over-expression system. Taurocholate flux was measured across human apical sodium-dependent bile acid transporter (hASBT)-Madin-Darby canine kidney monolayers on different occasions and kinetic parameters estimated with and without considering ABL. In error-free simulation/regression studies, flux values were generated across a range of J max, Kt, and substrate concentrations. Similar evaluation was performed for transport inhibition studies. Additionally, simulation/regression studies were performed, incorporating 15% random error to estimate the probability of successfully estimating Kt. Across different occasions, experimental J max and Kt estimates for taurocholate were strongly associated (p < 0.001; r2 = 0.82) when ABL was not considered. Simulation/regression results indicate that not considering ABL caused this association, such that Kt estimates were highly positively biased at high hASBT expression. In reanalyzing taurocholate flux data using the ABL-present model, Kt was relatively constant across occasions (approximately 5 microM) and not associated with J max (p = 0.24; r2 = 0.13). Simulations suggest that J max and Kt collectively determined ABL influence, which is most prominent under conditions of low monolayer resistance. Additionally, not considering ABL lead to negatively biased Ki estimates, especially at high J max. Error-inclusive simulation/regression studies indicated that the probability of successfully estimating Kt depended on the contribution of ABL resistance to flux; when flux became increasingly ABL-limited, probability of success decreased. Results indicate that ABL resistance can bias Kt and Ki estimates from overexpression systems, where the extent of bias is determined by transporter expression level and substrate affinity.     

Sister Mary Jospeh’s nodule as initial presentation of carcinoma caecum-case report and literature review

Umbilical metastases [Sister Mary Joseph’s nodules (SMJN)] are relatively rare and are associated usually with advanced intra-abdominal tumors from the gastrointestinal tract (GIT) or from the gynecological malignancies and they carry poor prognosis. Here we report a case of carcinoma caecum whose initial presentation was with umbilical metastases and a review of literature in relation to the umbilical metastases from colonic tumors.     

A Comparative Study on Surgical Outcomes of Glued Intraocular Lens and Sutured Scleral Fixated Intraocular Lens Implantation

Purpose: To compare the surgical outcomes of glued intraocular lens (GIOL) and sutured scleral fixated intraocular lens (SFIOL) implantation. Methods: A retrospective, interventional case series of 60 patients (68 eyes) who underwent GIOL (28 eyes: 11 primary; 17 secondary) and SFIOL (40 eyes: 24 primary; 16 secondary) in a tertiary eye care institute was done. The main outcome measures were complications, time taken for surgery, postoperative refractive outcomes, and final best-corrected visual acuity (BCVA). Results: The mean final BCVA was 0.42 (20/40) (+/– SD 0.29) in the GIOL group and 0.57 (20/50) (+/– SD 0.48) in the SFIOL group (p=0.08). The time taken for primary GIOL (mean+/– SD: 66+/–19 minutes) was significantly lower than primary SFIOL (means+/–SD: 94+/–34 minutes) (p=0.006). The complications were comparable between the GIOL (25%) and SFIOL (15%) groups (p=0.30). The most common complication noted was transient vitreous hemorrhage (GIOL 17%, SFIOL 5%). The only sight-threatening complication seen was retinal detachment, with one eye in each group. The mean follow-up was 20 (GIOL) and 20.4 weeks (SFIOL). Conclusion: Both glued intraocular lens and sutured scleral fixated intraocular lens are safe and effective procedures in cases of insufficient capsular support. In view of the shorter surgical time with similar outcomes, primary GIOL can be considered as a preferred surgical option over primary SFIOL.