Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia

Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.     

NAAG Peptidase Inhibition in the Periaqueductal Gray and Rostral Ventromedial Medulla Reduces Flinching in the Formalin Model of Inflammation

ABSTRACT: BACKGROUND: Metabotropic glutamate receptors (mGluRs) have been identified as significant analgesic targets. Systemic treatments with inhibitors of the enzymes that inactivate the peptide transmitter N-acetylaspartylglutamate (NAAG), an mGluR3 agonist, have an analgesia-like effect in rat models of inflammatory and neuropathic pain. The goal of this study was to begin defining locations within the central pain pathway at which NAAG activation of its receptor mediates this effect. RESULTS: NAAG immunoreactivity was found in neurons in two brain regions that mediate nociceptive processing, the periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM). Microinjection of the NAAG peptidase inhibitor ZJ43 into the PAG contralateral, but not ipsilateral, to the formalin injected footpad reduced the rapid and slow phases of the nociceptive response in a dose-dependent manner. ZJ43 injected into the RVM also reduced the rapid and slow phase of the response. The group II mGluR antagonist LY341495 blocked these effects of ZJ43 on the PAG and RVM. NAAG peptidase inhibition in the PAG and RVM did not affect the thermal withdrawal response in the hot plate test. Footpad inflammation also induced a significant increase in glutamate release in the PAG. Systemic injection of ZJ43 increased NAAG levels in the PAG and RVM and blocked the inflammation-induced increase in glutamate release in the PAG. CONCLUSION: These data demonstrate a behavioral and neurochemical role for NAAG in the PAG and RVM in regulating the spinal motor response to inflammation and that NAAG peptidase inhibition has potential as an approach to treating inflammatory pain via either the ascending (PAG) and/or the descending pain pathways (PAG and RVM) that warrants further study.     

Expanded mutational spectrum of the GLI3 gene substantiates genotype–phenotype correlations

Greig cephalopolysyndactyly syndrome (GCPS) and isolated preaxial polydactyly type IV (PPD-IV) are rare autosomal dominant disorders, both caused by mutations in the GLI3 gene. GCPS is mainly characterised by craniofacial abnormalities (macrocephaly/prominent forehead, hypertelorism) and limb malformations, such as PPD-IV, syndactyly and postaxial polydactyly type A or B (PAPA/B). Mutations in the GLI3 gene can also lead to Pallister?CHall syndrome (PHS) and isolated PAPA/B. In this study, we investigated 16 unrelated probands with the clinical diagnosis of GCPS/PPD-IV and found GLI3 mutations in 12 (75?%) of them (nine familial and three sporadic cases). We also performed a detailed clinical evaluation of all 12 GLI3-positive families, with a total of 27 patients. The hallmark triad of GCPS (preaxial polydactyly, macrocephaly/prominent forehead, hypertelorism) was present in 14 cases (52?%), whereas at least one typical dysmorphic feature was manifested in 17 patients (63?%). Upon sequencing of the GLI3 gene, we demonstrated eight novel and two previously reported heterozygous point mutations. We also performed multiplex ligation-dependent probe amplification (MLPA) to screen for intragenic copy number changes and identified heterozygous deletions in the two remaining cases (16.7?%). Our findings fully support previous genotype?Cphenotype correlations, showing that exonic deletions, missense mutations, as well as truncating variants localised out of the middle third of the GLI3 gene result in GCPS/PPD-IV and not PHS. Additionally, our study shows that intragenic GLI3 deletions may account for a significant proportion of GCPS/PPD-IV causative mutations. Therefore, we propose that MLPA or quantitative polymerase chain reaction (qPCR) should be implemented into routine molecular diagnostic of the GLI3 gene.     

Retrospective analysis of idiopathic scoliosis medical records coming from one out-patient clinic for compatibility with Scoliosis Research Society criteria for brace treatment studies

Background

First author attempted to analyse medical records of patients with idiopathic scoliosis for compliance with the Scoliosis Research Society brace studies criteria. A retrospective analysis of medical records of 2705 girls treated from 1989 to 2002 was carried out.

Methods

Age, Cobb, Risser and menarchal status were analyzed for compliance with the Scoliosis Research Society brace studies criteria: a) age ≥10 years, b) Risser 0–2, c) 25–40° Cobb angle, d) no earlier treatment, e) patients before first menses or not more than one year from first menses.

Results

It has been found that 183 girls out of 2705 were ≥10 years old and in the range 25–40° Cobb angle. One hundred two out of 2705 patients revealed eligible for brace effectiveness study according to SRS 2005 criteria. 120 out of 2705 patients revealed eligible for brace brace effectiveness study according to SRS-SOSORT 2014 criteria.

Conclusion

The excluded patients revealed too old or with too significant Cobb angles. This indicates the changing criteria for scoliosis brace treatment over the time. Direct comparison of current results of brace treatment with historical series of cases turns out to be very difficult.

     

Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element

The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27 gene promoter. The conformational changes in the protein-DNA during the formation of the UspDBD-EcRDBD-hsp27 complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5'- and/or 3'-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5'-end of the regulatory element and moved close to the 3'-end. An analysis was also done on the effect that DHR38DBD, the Drosophila ortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27 bending was induced by DHR38DBD. Fluorescence data revealed that hsp27 had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27 in the presence of EcRDBD is presented.     

Inflammatory bowel disease - is there something new in the immunological background?

In the present paper we correlate clinical data, as well as histopathological, immunohistochemical and molecular biology methods, with the occurrence of both forms of inflammatory bowel disease (IBD) i.e. ulcerative colitis and Crohn's disease. We found that patients with a history of Epstein-Barr virus (EBV) or cytomegalovirus (CMV) infections, as well as steroid treatment, had increased susceptibility to the development of IBD. The diagnosis of IBD was confirmed by histopathology. Previous infections by EBV and CMV, as well as M. tuberculosis, were proved by PCR-based techniques and in situ hybridization. We found PCR-proved latent viral infections in 30-50% of the IBD patients we studied. However, we were unable to prove the presence of viral antigens by immunohistochemistry for EBV or CMV. We found positive correlations between the presence of anti-CMV IgG, as well as PCR-positive results for M. tuberculosis with an ulcerative colitis diagnosis. Additionally, up to 80% of IBD patients used steroids, which was found to be correlated with a diagnosis of Crohn's disease. Our data may support the theory that IBD could be related to previous viral infections and the use of steroids.     

Molecular cross-talk between the NRF2/KEAP1 signaling pathway, autophagy, and apoptosis

Oxidative stress, perturbations in the cellular thiol level and redox balance, affects many cellular functions, including signaling pathways. This, in turn, may cause the induction of autophagy or apoptosis. The NRF2/KEAP1 signaling pathway is the main pathway responsible for cell defense against oxidative stress and maintaining the cellular redox balance at physiological levels. The relation between NRF2/KEAP1 signaling and regulation of apoptosis and autophagy is not well understood. In this hypothesis article we discuss how KEAP1 protein and its direct interactants (such as PGAM5, prothymosin α, FAC1 (BPTF), and p62) provide a molecular foundation for a possible cross-talk between NRF2/KEAP1, apoptosis, and autophagy pathways. We present a hypothesis for how NRF2/KEAP1 may interfere with the cellular apoptosis-regulatory machinery through activation of the ASK1 kinase by a KEAP1 binding partner-PGAM5. Based on very recent experimental evidence, new hypotheses for a cross-talk between NF-κB and the NRF2/KEAP1 pathway in the context of autophagy-related "molecular hub" protein p62 are also presented. The roles of KEAP1 molecular binding partners in apoptosis regulation during carcinogenesis and in neurodegenerative diseases are also discussed.     

Large differences in peak oxygen uptake do not independently alter changes in core temperature and sweating during exercise

The independent influence of peak oxygen uptake (Vo(? peak)) on changes in thermoregulatory responses during exercise in a neutral climate has not been previously isolated because of complex interactions between Vo(? peak), metabolic heat production (H(prod)), body mass, and body surface area (BSA). It was hypothesized that Vo(? peak) does not independently alter changes in core temperature and sweating during exercise. Fourteen males, 7 high (HI) Vo(? peak): 60.1 ± 4.5 ml·kg?1·min?1; 7 low (LO) Vo(? peak): 40.3 ± 2.9 ml·kg?1·min?1 matched for body mass (HI: 78.2 ± 6.1 kg; LO: 78.7 ± 7.1 kg) and BSA (HI: 1.97 ± 0.08 m2; LO: 1.94 ± 0.08 m2), cycled for 60-min at 1) a fixed heat production (FHP trial) and 2) a relative exercise intensity of 60% Vo(? peak) (REL trial) at 24.8 ± 0.6°C, 26 ± 10% RH. In the FHP trial, H(prod) was similar between the HI (542 ± 38 W, 7.0 ± 0.6 W/kg or 275 ± 25 W/m2) and LO (535 ± 39 W, 6.9 ± 0.9 W/kg or 277 ± 29 W/m2) groups, while changes in rectal (T(re): HI: 0.87 ± 0.15°C, LO: 0.87 ± 0.18°C, P = 1.00) and aural canal (T(au): HI: 0.70 ± 0.12°C, LO: 0.74 ± 0.21°C, P = 0.65) temperature, whole-body sweat loss (WBSL) (HI: 434 ± 80 ml, LO: 440 ± 41 ml; P = 0.86), and steady-state local sweating (LSR(back)) (P = 0.40) were all similar despite relative exercise intensity being different (HI: 39.7 ± 4.2%, LO: 57.6 ± 8.0% Vo(2 peak); P = 0.001). At 60% Vo(2 peak), H(prod) was greater in the HI (834 ± 77 W, 10.7 ± 1.3 W/kg or 423 ± 44 W/m2) compared with LO (600 ± 90 W, 7.7 ± 1.4 W/kg or 310 ± 50 W/m2) group (all P < 0.001), as were changes in T(re) (HI: 1.43 ± 0.28°C, LO: 0.89 ± 0.19°C; P = 0.001) and T(au) (HI: 1.11 ± 0.21°C, LO: 0.66 ± 0.14°C; P < 0.001), and WBSL between 0 and 15, 15 and 30, 30 and 45, and 45 and 60 min (all P < 0.01), and LSR(back) (P = 0.02). The absolute esophageal temperature (T(es)) onset for sudomotor activity was ～0.3°C lower (P < 0.05) in the HI group, but the change in T(es) from preexercise values before sweating onset was similar between groups. Sudomotor thermosensitivity during exercise were similar in both FHP (P = 0.22) and REL (P = 0.77) trials. In conclusion, changes in core temperature and sweating during exercise in a neutral climate are determined by H(prod), mass, and BSA, not Vo(? peak).     

Probing Orientational Behavior of MHC Class I Protein and Lipid Probes in Cell Membranes by Fluorescence Polarization-Resolved Imaging

Steady-state polarization-resolved fluorescence imaging is used to analyze the molecular orientational order behavior of rigidly labeled major histocompatibility complex class I (MHC I) proteins and lipid probes in cell membranes of living cells. These fluorescent probes report the orientational properties of proteins and their surrounding lipid environment. We present a statistical study of the molecular orientational order, modeled as the width of the angular distribution of the molecules, for the proteins in the cell endomembrane and plasma membrane, as well as for the lipid probes in the plasma membrane. We apply this methodology on cells after treatments affecting the actin and microtubule networks. We find in particular opposite orientational order changes of proteins and lipid probes in the plasma membrane as a response to the cytoskeleton disruption. This suggests that MHC I orientational order is governed by its interaction with the cytoskeleton, whereas the plasma membrane lipid order is governed by the local cell membrane morphology.