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Nature 606(7915), 678 - 682 (2022). doi:10.1038/s41586-022-04827-6, Published by Nature Publ. Group, London [u.a.]

Background: Ductal carcinoma in situ (DCIS) is a risk factor for the development of invasive ductal carcinoma. Previous work has shown that an increased presence of T cells corresponds to high-risk DCIS features: age 5 cm, high-grade, palpable mass, hormone receptor negativity, and HER2 positivity. The presence of T cells colocalized with tumor is emerging as a predictor of response to checkpoint inhibition in invasive cancer. We previously have demonstrated that intratumoral injections of a checkpoint inhibitor (pembrolizumab) in DCIS resulted in an increased number of T cells, but without tumor cell killing. We hypothesized that the addition of an agent that could potentiate both innate and adaptive immune responses might increase the efficacy of checkpoint inhibition. Methods/Materials: We administered intralesional injections of immunotherapy (4-8 mg pembrolizumab + 2-4mg mRNA 2752) to patients with “high-risk” DCIS in an attempt to modulate the immune system and protect from disease progression and recurrence. Two patients have been enrolled and completed therapy and 4 more are being treated. Immune infiltrates were characterized by multiplex immunofluorescence (mIF) staining of pre- and post-treatment tissue samples using two 6-plex panels. Staining was performed on a Leica Bond autostainer and images were acquired on a Vectra Polaris imaging system. Image analysis was performed utilizing inForm and QuPath software packages, as well as R scripts. Results: The first patient treated, a 41-year-old woman with ER+/PR+, HER2- disease, with low T cells in her pre-treatment biopsy, received one dose of pembrolizumab (8mg) with mRNA 2752 (4mg). While she experienced fatigue, sustained fever, and muscle aches for 3-5 days, draining node enlargement and redness and swelling at the breast a week after treatment, there was no change on her post-treatment MRI, and no significant change in the number of T cells in her post-treatment tissue. The second patient, a 74-year-old woman with ER-/PR-, HER2+ disease had high numbers of T cells in the pre-treatment biopsy. She received one injection of pembrolizumab (4mg) with mRNA 2752 (2mg), followed 3 weeks later by an injection of pembrolizumab (4mg) alone. She declined the second dose of mRNA 2752 due to side effects from the first injection. Her post-treatment MRI showed some reduction in tumor volume and her post-treatment biopsy showed no evidence of DCIS, as well as an increase in T cells. Conclusions: The presence of T cells prior to therapy is likely to be important for eliciting a strong anti-tumor response. The addition of an agent that enhances both innate and adaptive immune responses may increase the efficacy of immune checkpoint blockade. Four additional patients will have completed their therapy by the end of August and we await results. It will be important to understand if pre-existing T cells within the DCIS lesion are a predictor for response to therapy as we continue to screen and enroll patients. Citation Format: Nicole F. Schindler, Alexa Glencer, Phoebe Miller, Christopher Schwartz, Gillian Hirst, Alexander Borowsky, Michael Campbell, Laura Esserman. Intralesional injections with checkpoint inhibitors and cytokines can modify DCIS lesions with infiltrating T cells [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B005.

Background/Aim: Testing for human epidermal growth factor receptor 2 (HER2) positivity in women diagnosed with ductal carcinoma in situ (DCIS) is not performed routinely, although the frequency of HER2+ DCIS is at least 30%. HER2+ DCIS lesions may be at higher risk of progression to invasive carcinoma, and specific isoforms of HER2 may lead to different malignant potential. Loss of the HER2 extracellular domain engineered in mouse models demonstrates rapid progression to invasive behavior. Evaluating isoforms of HER2 may aid in classifying DCIS lesions as higher or lower risk, and may provide insight for novel treatment and prevention approaches. Methods: A cohort of high risk DCIS cases were assembled, enriched for the presence of high risk features, including large size (>5 cm) and palpable. Immunohistochemistry (IHC) and FISH were used to identify cases that were HER2 positive. MultiplexIHC was used to detect extracellular and intracellular domains of HER2, with imaging analysis performed to identify HER2 isoforms of HER2+ specimens. Isoforms were characterized as 1) full-length/extracellular domain (ECD) intact, 2) pure/complete loss of ECD (p95 isoform), 3) subclonal populations of both full-length ECD and p95 and 4) gradient/representing partial loss of ECD per cell (reflecting post-translational cleavage). Results: Out of 51 cases, 34 (67%) cases were HER2+. In multiplexIHC analysis for HER2, 51% displayed gradient (partial) or focal (subclonal) loss of the extracellular domain. 33% displayed pure p95 isoform (complete loss of ECD). Only 16% of the cohort displayed full-length HER2 extracellular domain. Conclusions: HER2 expression is much more common in large in situ lesions than in invasive lesions. This suggests that HER2 positivity may play a role in protecting against invasion. Investigation of HER2 Isoform testing using multiplex staining reveals heterogeneity with partial or complete loss of the HER2 extracellular domain being most common. Loss of the extracellular domain of human epidermal growth factor receptor 2 (HER2) p95 isoform is predominant among patients diagnosed with HER2 positive ductal carcinoma in situ (DCIS). Citation Format: Nicole F. Schindler, Alexa Glencer, Hidetoshi Mori, Gillian Hirst, Cristian Maldonado Rodas, Michael Campbell, Laura Esserman, Alexander Borowsky. Loss of the extracellular domain of human epidermal growth factor receptor 2 (HER2) p95 isoform is common in large DCIS lesions [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B013.

The tetraneutron has attracted the attention of nuclear physicists during the past decades, but there is still no unambiguous confirmation of its existence or non-existence. A new experiment based on $$^{8}$$ He(p, 2p) $$^{7}$$ H{t+ $$^{4}n$$ } reaction, with direct detection of the four neutrons, has been carried out at RIBF, which can hopefully help to draw a definite conclusion on the tetraneutron system.

Nuclear instruments & methods in physics research / A 1014, 165701 (2021). doi:10.1016/j.nima.2021.165701, Published by North-Holland Publ. Co., Amsterdam

Neurons have recently emerged as essential cellular constituents of the tumour microenvironment, and their activity has been shown to increase the growth of a diverse number of solid tumours1. Although the role of neurons in tumour progression has previously been demonstrated2, the importance of neuronal activity to tumour initiation is less clear—particularly in the setting of cancer predisposition syndromes. Fifteen per cent of individuals with the neurofibromatosis 1 (NF1) cancer predisposition syndrome (in which tumours arise in close association with nerves) develop low-grade neoplasms of the optic pathway (known as optic pathway gliomas (OPGs)) during early childhood3,4, raising the possibility that postnatal light-induced activity of the optic nerve drives tumour initiation. Here we use an authenticated mouse model of OPG driven by mutations in the neurofibromatosis 1 tumour suppressor gene (Nf1)5 to demonstrate that stimulation of optic nerve activity increases optic glioma growth, and that decreasing visual experience via light deprivation prevents tumour formation and maintenance. We show that the initiation of Nf1-driven OPGs (Nf1-OPGs) depends on visual experience during a developmental period in which Nf1-mutant mice are susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly increased shedding of neuroligin 3 (NLGN3) within the optic nerve in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacological inhibition of NLGN3 shedding blocks the formation and progression of Nf1-OPGs. Collectively, our studies establish an obligate role for neuronal activity in the development of some types of brain tumours, elucidate a therapeutic strategy to reduce OPG incidence or mitigate tumour progression, and underscore the role of Nf1mutation-mediated dysregulation of neuronal signalling pathways in mouse models of the NF1 cancer predisposition syndrome. Mouse models of NF1-associated optic pathway glioma show that tumour initiation and growth are driven by aberrantly high levels of NLGN3 shedding in the optic nerve in response to retinal neuron activity.

The isospin characters of low-energy dipole excitations in neutron-rich unstable nucleus 20O were investigated, for the first time in unstable nuclei. Two spectra obtained from a dominant isovector probe (View the MathML sourceO20+Au) and a dominant isoscalar probe (View the MathML sourceO20+α) were compared and analyzed by the distorted-wave Born approximation to extract independently the isovector and isoscalar dipole strengths. Two known 1−1− states with large isovector dipole strengths at energies of 5.36(5) MeV (View the MathML source11−) and 6.84(7) MeV (View the MathML source12−) were also excited by the isoscalar probe. These two states were found to have different isoscalar dipole strengths, 2.70(32)% (View the MathML source11−) and 0.67(12)% (View the MathML source12−), respectively, in exhaustion of the isoscalar dipole-energy-weighted sum rule. The difference in isoscalar strength indicated that they have different underlying structures. ispartof: Physics Letters B vol:768 pages:387-392 status: published

7 pags., 3 figs. -- 27th International Nuclear Physics Conference (INPC2019), The tetraneutron has attracted the attention of nuclear physicists during the past decades, but there is still no unambiguous confirmation of its existence or nonexistence. A new experiment based on 8 He(p,2p)7H{t+4n} reaction, with direct detection of the four neutrons, has been carried out at RIBF, which can hopefully help to draw a definite conclusion on the tetraneutron system., We acknowledge the support of the RIBF accelerator staff and the BigRIPS team for providing the high-quality beam. Z. H. Yang acknowledges the financial support from the Foreign Postdoctoral Researcher program of RIKEN. T. Aumann acknowledges the support by DFG via SFB 1245. P. Koseoglou acknowledges the support from BMBF (NUSTAR.DA grant No.05P 15RDFN1).

Spectroscopic factors of neutron-hole and proton-hole states in ^{131}Sn and ^{131}In, respectively, were measured using one-nucleon removal reactions from doubly magic ^{132}Sn at relativistic energies. For ^{131}In, a 2910(50)-keV γ ray was observed for the first time and tentatively assigned to a decay from a 5/2^{-} state at 3275(50) keV to the known 1/2^{-} level at 365 keV. The spectroscopic factors determined for this new excited state and three other single-hole states provide first evidence for a strong fragmentation of single-hole strength in ^{131}Sn and ^{131}In. The experimental results are compared to theoretical calculations based on the relativistic particle-vibration coupling model and to experimental information for single-hole states in the stable doubly magic nucleus ^{208}Pb.

Distilled water plus 0.1% surfactant suspensions of spores of Aspergillus flavus and Aspergillus parasiticus were exposed to several radiation levels of cobalt-60 gamma rays. Spores of A. flavus isolate M-141 were exposed to radiation levels of approximately 16, 90 and 475 Krads and inoculated onto a sterile rice substrate which was then monitored for aflatoxin production. In this initial trial with A. flavus M-141, aflatoxins B1 and M production on rice increased as radiation dose increased. At the highest dose, this increase was more than 50 times higher than the non-irradiated controls. Spores of an aflatoxin G1-producing A. parasiticus isolate, M-1094, were exposed to 90, 215 and 430 Krads and resulted in increased production of aflatoxins G1, B1, and M. Aflatoxin production by M-1094 was highest at the low and medium dose levels. Irradiation of spores of this isolate with 430 Krads produced no observable spore germination or growth on rice and no detectable aflatoxin after 1 week of incubation at 27 ...

Neurons have recently emerged as essential cellular constituents of the tumor microenvironment, where their activity increases the growth of a diverse number of solid tumors. While the role of neurons in tumor progression has been previously demonstrated, the importance of neuronal activity to tumor initiation is less clear, particularly in the setting of cancer predisposition syndromes. In the Neurofibromatosis type 1 (NF1) cancer predisposition syndrome, in which tumors arise in close association with nerves, 15% of individuals develop low-grade neoplasms of the optic pathway (optic gliomas) during early childhood, raising the intriguing possibility that postnatal light-induced optic nerve activity drives tumor initiation. Here, we employ an authenticated murine model of Nf1 optic glioma to demonstrate that stimulation of optic nerve activity increases optic glioma growth, while decreasing optic nerve activity via light deprivation prevents tumor formation and maintenance. By manipulating environmental light to modulate optic pathway (retinal) neuron activity, we show that Nf1 optic glioma initiation depends on neuronal activity during a developmental period susceptible to tumorigenesis. Germline Nf1 mutation in retinal neurons results in aberrantly high optic nerve neuroligin-3 (Nlgn3) shedding in response to retinal neuronal activity. Moreover, genetic Nlgn3 loss or pharmacologic inhibition of Nlgn3 shedding blocks murine Nf1 optic gliomagenesis and progression. Collectively, these studies establish an obligate role for neuronal activity in the development of certain brain tumors, elucidate a therapeutic strategy to reduce optic glioma incidence or mitigate tumor progression, and underscore the role of Nf1 mutation-mediated dysregulation of neuronal signaling pathways in the NF1 cancer predisposition syndrome.

The tetraneutron has been drawing the attention of the nuclear physics community for decades, but a firm conclusion on its existence and properties is still far from being reached despite many experimental and theoretical efforts. New measurements have recently been performed at RIBF with the SAMURAI spectrometer by applying complementary reaction probes, which will help to pin down the properties of this four-neutron system., Comment: 5 pages, 2 figures, conference proceedings of XXII International Conference on Few-Body Problems in Physics (FB22), Caen, France, July 2018

Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE(-/-) mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC50 value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance.

The species of the family Goodeidae have evolved reproductive strategies involving intraovarian gestation, early evacuation of nearly yolk-exhausted embryos from the ovigerous tissue into the ovarian cavity, placental matrotrophy during intraluminal gestation, and the birth of highly developed fry. The inner ovarian lining becomes hypervascularized during gestational periods and functions as the maternal component of the placental association. Embryotrophic liquid is secreted by the inner ovarian epithelium into the ovarian cavity. Comparative electrophoretic analyses of embryotrophe and maternal blood serum provide evidence for the transfer of maternal serum proteins into the embryotrophe. Trophotaeniae, proctodaeal processes of the embryos, provide a surface for nutrient absorption. Endocytic activity was demonstrated by ingestion of unspecific tracer proteins in various species. Moreover, the trophotaenial absorptive cells (TACs) in Ameca splendens ingest various proteins or random copolymers conjugated to colloidal gold as well as radioiodinated proteins in a way that satisfies the criteria of receptor-mediated endocytosis. Several aminopeptidases (APs) on the surface of TACs were identified as protein binding sites as evidenced by inhibition of binding and uptake of marker proteins in the presence of AP substrates or AP inhibitors. Morphological adaptations of the embryonic circulatory system pertaining to nutrient and gas exchange were characterized. The embryonic epidermis comprises two layers of squamous cells closely underlain by a dense capillary net. Efficient gas exchange is facilitated by a thin embryotrophe-blood barrier of both the embryonic skin and the intraovarian lining.

Paullinia cupana (guaraná) is an Amazonian plant that has been previously shown to be effective in treating chemotherapy-related fatigue (CRF) in patients with breast cancer. We aimed to evaluate the efficacy of a purified dry extract of P. cupana (PC-18) in patients with various solid tumors treated with chemotherapy.We included 40 patients with solid tumors who showed increases in their Brief Fatigue Inventory (BFI) questionnaire scores after 1 week of systemic chemotherapy. PC-18 was administered at 37.5 mg by mouth two times per day (PO bid), starting after 1 week of chemotherapy, for 3 weeks (induction phase). Patients who had an improvement in or stabilization of their BFI scores were randomized to receive either PC-18 at the same dose or placebo for the following 3 weeks (maintenance phase).After PC-18 treatment, the BFI fatigue scores improved or stabilized in 36 out of the 40 patients (mean BFI score difference = 2.503; 95% confidence interval: 1.716-3.375, p = .0002). Three weeks after randomization (16 patients on PC-18 and 17 on placebo), we observed no significant differences in the BFI, Functional Assessment of Chronic Illness Therapy, Hospital Anxiety and Depression Scale, and Pittsburgh Sleep Quality Index scores between patients randomized to PC-18 versus placebo.We conclude that the PC-18 extract may be effective for the treatment of CRF in patients with a variety of solid tumors. A conditioning effect, which was observed in patients who had a beneficial effect of PC-18 on CRF, may explain the better than expected fatigue scores of the placebo-treated patients.

A series of N-aryl pyridinone inhibitors of p38 mitogen activated protein (MAP) kinase were designed and prepared based on the screening hit SC-25028 (1) and structural comparisons to VX-745 (5). The focus of the investigation targeted the dependence of potency and metabolic stability on the benzyloxy connectivity, the role of the C-6 position and the substitution pattern on the N-phenyl ring. Further optimization produced the highly selective and potent pyridinones 2 and 3. These inhibitors exhibited activity in both acute and chronic models of inflammation.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-dependent enzyme that catalyzes the initial and rate-limiting step of tryptophan catabolism resulting in the local depletion of tryptophan and the concomitant production of kynurenine, both of which are immunosuppressive. Targeting IDO1 in combination with PD-1/PD-L1-targeted antibodies has shown promise in early phase clinical trials in several cancers and strongly suggests that, in some patients, IDO1 expression restrains PD-1/PD-L1-targeted checkpoint therapies. While some cancers extrinsically express IDO1 in response to IFN-γ produced from an ongoing, yet ineffective immune response, others select for the intrinsic expression of IDO1, independent of an immune response. We identified several cancer cell lines that intrinsically expressed either IDO1 or the related isozyme TDO2. Using these cell lines, we discovered LY3381916, a potent and selective inhibitor of cell-based IDO1 activity (IDO1 7 nM; TDO2 >20 µM). Using a variety of techniques, we demonstrated that LY3381916 binds to newly synthesized apo-IDO1 lacking heme, but does not inhibit mature heme-bound IDO1. Protein x-ray crystallography confirmed that LY3381916 binds to apo-IDO1 where it occupies the heme-binding pocket of IDO1. As a result of this novel mechanism of action, substantial inhibition of IDO1 in tumors requires the turn-over of mature heme-bound IDO1. Modeling of the pre-clinical PK/PD relationship suggests QD dosing of LY3381916 will maintain greater than 90% inhibition over 24 hours. In addition, due to the favorable properties of the drug, significant central nervous system (CNS) penetration has been measured for LY3381916 (rodent kp,uu 0.26). Kynurenine-mediated agonism of the aryl hydrocarbon receptor (AHR) is immunosuppressive in the tumor microenvironment. Inhibition of IDO1 and the subsequent reduction of kynurenine can relieve this immunosuppression. However, several heme-binding IDO1 inhibitors have been shown to replace kynurenine as an AHR agonist potentially limiting their ability to relieve this IDO1-dependent immunosuppressive mechanism. LY3381916 shows no confounding agonism of AHR up to 100 µM. Additionally, we characterized LY3381916 in pre-clinical tumor models and demonstrated that it was able to enhance LY3300054, anti-PD-L1 antibody (LY3300054) activity, which was associated with an enhanced T cell response. Based on these characteristics, LY3381916 is currently being investigated in a Phase I clinical trial. These data suggest further development of LY3381916 may be warranted. Citation Format: Frank C. Dorsey, Karim A. Benhadji, Lillian L. Sams, Debra A. Young, John F. Schindler, Karen L. Huss, Alexander Nikolayev, Carmine Carpenito, David Clawson, Bonita Jones, Andrew L. Faber, James E. Thomas, Steven A. Haney, Gaiying Zhao, William T. McMillen, Tod Smeal, Daniel J. Sall, Michael D. Kalos, Sandaruwan Geeganage, James R. Henry. Identification and characterization of the IDO1 inhibitor LY3381916 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5245.

Activation of the p38 kinase pathway in immune cells leads to the transcriptional and translational regulation of proinflammatory cytokines. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), a direct downstream substrate of p38 kinase, regulates lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) production through modulating the stability and translation of these mRNAs. Developing small-molecule inhibitors of MK2 may yield anti-inflammatory efficacy with a different safety profile relative to p38 kinase inhibitors. This article describes the pharmacologic properties of a benzothiophene MK2 inhibitor, PF-3644022 [(10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8H-[1,4]diazepino[5',6':4,5]thieno[3,2-f]quinolin-8-one]. PF-3644022 is a potent freely reversible ATP-competitive compound that inhibits MK2 activity (K(i) = 3 nM) with good selectivity when profiled against 200 human kinases. In the human U937 monocytic cell line or peripheral blood mononuclear cells, PF-3644022 potently inhibits TNFalpha production with similar activity (IC(50) = 160 nM). PF-3644022 blocks TNFalpha and IL-6 production in LPS-stimulated human whole blood with IC(50) values of 1.6 and 10.3 microM, respectively. Inhibition of TNFalpha in U937 cells and blood correlates closely with inhibition of phospho-heat shock protein 27, a target biomarker of MK2 activity. PF-3644022 displays good pharmacokinetic parameters in rats and is orally efficacious in both the rat acute LPS-induced TNFalpha model and the chronic streptococcal cell wall-induced arthritis model. Dose-dependent inhibition of TNFalpha production in the acute model and inhibition of paw swelling in the chronic model is observed with ED(50) values of 6.9 and 20 mg/kg, respectively. PF-3644022 efficacy in the chronic inflammation model is strongly correlated with maintaining a C(min) higher than the EC(50) measured in the rat LPS-induced TNFalpha model.

Janus-associated kinases (JAKs) play critical roles in cytokine signaling, and have emerged as viable therapeutic targets in inflammation and oncology related diseases. To date, targeting JAK proteins with highly selective inhibitor compounds have remained elusive. We have expressed the active kinase domains for both JAK2 and JAK3 and devised purification protocols to resolve the non-, mono- (Y1007) and diphosphorylated (Y1007 and Y1008) states of JAK2 and non- and monophosphorylated states of JAK3 (Y980). An optimal purified protein yield of 20, 29 and 69mg per 20L cell culture was obtained for the three JAK2 forms, respectively, and 12.2 and 2.3mg per 10L fermentation for the two JAK3 forms allowing detailed biochemical and biophysical studies. To monitor the purification process we developed a novel HPLC activity assay where a sequential order of phosphorylation was observed whereby the first tyrosine residue was completely phosphorylated prior to phosphorylation of the tandem tyrosine residue. A Caliper-based microfluidics assay was used to determine the kinetic parameters (K(m) and k(cat)) for each phosphorylated state, showing that monophosphorylated (Y1007) JAK2 enzyme activity increased 9-fold over that of the nonphosphorylated species, and increased an additional 6-fold for the diphosphorylated (Y1007/Y1008) species, while phosphorylation of JAK3 resulted in a negligible increase in activity. Moreover, crystal structures have been generated for each isolated state of JAK2 and JAK3 with resolutions better than 2.4A. The generation of these reagents has enabled kinetic and structural characterization to inform the design of potent and selective inhibitors of the JAK family.

Nuclear factor (NF)-kappaB activation has been clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The central role that IkappaB kinase-2 (IKK-2) plays in regulating NF-kappaB signaling in response to inflammatory stimuli has made this enzyme an attractive target for therapeutic intervention. Although diverse chemical classes of IKK-2 inhibitors have been identified, the binding kinetics of these inhibitors has limited the scope of their applications. In addition, safety assessments of IKK-2 inhibitors based on a comprehensive understanding of the pharmacokinetic/pharmacodynamic relationships have yet to be reported. Here, we describe a novel, potent, and highly selective IKK-2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide]. PHA-408 is an ATP-competitive inhibitor, which binds IKK-2 tightly with a relatively slow off rate. In arthritis-relevant cells and animal models, PHA-408 suppresses inflammation-induced cellular events, including IkappaBalpha phosphorylation and degradation, p65 phosphorylation and DNA binding activity, the expression of inflammatory mediators, and joint pathology. PHA-408 was efficacious in a chronic model of arthritis with no adverse effects at maximally efficacious doses. Stemming from its ability to bind tightly to IKK-2, as a novelty, we demonstrated that PHA-408-mediated inhibition of IKK-2 activity correlated very well with its ability to modulate the fate of IKK-2 substrates and downstream transcriptional events. We ultimately directly linked IKK-2 activity ex vivo and in vivo to markers of inflammation with the inhibitor plasma concentrations. Thus, PHA-408 represents a powerful tool to further gain insight into the mechanisms by which IKK-2 regulates NF-kappaB signaling and validates IKK-2 as a therapeutic target.

Plus de 30 ans de recul de chirurgie fonctionnelle nous permettent d’affirmer aujourd’hui que pres de sept a huit patients tetraplegiques sur dix peuvent se voir proposer une chirurgie du ou des membres superieurs. La mise en route d’une collaboration multidisciplinaire dans ce domaine ne s’improvise pas. La reeducation postoperatoire doit s’appuyer sur des programmes codifies que des equipes aguerries ont eprouves. Le dialogue permanent etabli entre le chirurgien et l’equipe de medecine physique vise a repondre au souci de raccourcir les durees d’immobilisation tout en prevenant le risque de ruptures, d’adherences et d’autres complications locales.

La chirurgie fonctionnelle du membre superieur a plus de 40 ans de pratique et d’experience derriere elle (Ann Readapt Med Phys 46 (2003) 144–55). Les procedures sont codifiees, les techniques maitrisees. Les equipes aguerries a la reeducation de patients tetraplegiques operes savent que les annees d’experience se nourrissent d’un echange constant entre le chirurgien et les equipes de medecine physique et de readaptation et que la rigueur de la prise en charge postoperatoire reste de mise pour obtenir le meilleur resultat fonctionnel. Ce texte vise a introduire un numero special de La lettre de MPR qui sera consacre a ce sujet dans les prochains mois.

Affective mixed states in bipolar disorders are a current matter of scientific debate and represent a complex clinical picture with coexisting manic and depressive symptoms. Treatment of mixed states is regarded as an important challenge. As diagnostic uncertainties complicate systematic clinical evaluations of this patient group, generally accepted clinical treatment guidelines are lacking yet. In this review the significance as well as the problems and risks of new treatment options are discussed.

The conductor width dependence of the critical current density Jc in the grain boundary network occurring in RABiTS based coated conductors is simulated using experimentally obtained electron backscattering diffraction (EBSD) maps and the exponential dependence of the critical current density on the misorientation angle. It is found that a conductor width of around 20 grains (of average size) is sufficient to pass 90% of the maximum current density, independent of texture quality and grain aspect ratio in the current direction. The aspect ratio does however influence the absolute value of Jc, giving higher Jc values for higher aspect ratios. These results are in good agreement with Jc simulations based on purely statistical grain boundary distributions.

In the goodeid placental analogue, trophotaeniae provide extraembryonic gut-derived exchange surfaces. Ameca splendens embryos possess endocytosing trophotaeniae that are capable of absorbing a dazzling array of proteinaceous substances. The iron core protein, native ferritin (NF), and several radioiodinated proteinaceous substances were used to study ligand and binding site pathways in the trophotaenial absorptive cells (TACs). Time sequence analysis of NF trafficking indicated an exclusively lysosomal pathway. Binding to TACs of NF was completely inhibitable by proteins containing multiple lysine residues such as apoferritin, bovine serum albumin (BSA), human transferrin (HTf), fetuin, hemoglobin, myoglobin, cytochrome c, ubiquitin, parvalbumin as well as the random copolymers, poly(Glu,Lys,Tyr)6:3:1 and poly(D-Glu,D-Lys)6:4. Peptide hormones and pepsin that contains only one lysine residue did not produce inhibitory effects. Radiolabels such as 125I-BSA, 125I-HTf and 125I-poly(Glu,Lys,Tyr) bound to trophotaeniae in a specific saturable manner. Any two proteins were shown to hinder one another in getting hold of a binding site. Concentration-dependent 125I-BSA binding and Scatchard analysis of the data revealed both low- and medium-affinity binding with apparent dissociation constants, Kds, of 3.4×10−5 M and 2×10−7 M, respectively. Binding of NF and radioiodinated proteins was inhibited in the presence of a large excess of L-Lys, D-Lys, and several dipeptides containing Lys. Both Ca2+-depletion and low pH dramatically reduced the TACs' capacity to bind proteins. The effects of acidotropic agents included a reversible loss of surface protein binding sites, tremendous vacuolation, and the arrest of lysosomal degradation. Collectively, present results demonstrate that TACs bind and absorb multiple proteinaceous substances through a mechanism satisfying the criteria of receptor-mediated endocytosis. It is concluded that scavenger protein binding sites are used to ingest proteins for lysosomal degradation, helping to meet the embryos' amino acid requirement. J. Exp. Zool. 299A:197–212, 2003. © 2003 Wiley-Liss, Inc.

The grain boundary (GB) network in biaxially textured YBa/sub 2/Cu/sub 3/O/sub x/ (Y123) coated conductors is the key to their transport properties and determines the upper limit for the critical current density. In this contribution we report on detailed EBSD (electron backscattering diffraction) investigations of the formation of these GB networks in Y123 films deposited on highly biaxially textured metallic substrates and the comparison of the measured critical current density with simulations based on the exact knowledge of the underlying GB network. Highly cube textured Ni-tapes were prepared by rolling and recrystallization and detailed texture maps were determined by EBSD before and after the deposition of Y123 films by thermal evaporation or pulsed laser deposition. This allowed the quantitative evaluation of both the Y123-growth on different oriented Ni-grains and the GB misorientation network which determines the critical current density. This exact texture information was also used to calculate the critical current density based on the well known exponential j/sub c/-dependence across Y123 tilt GBs and is compared to the measured transport j/sub c/ across the same substrate area.

The binding of N-cyclohexylformamide (CXF) to the complex of horse liver alcohol dehydrogenase with NADH mimics that of the Michaelis complex for aldehyde reduction catalyzed by the enzyme. The Raman spectra of bound CXF and its 13C- and 15N-substituted derivatives have been obtained using Raman difference techniques, and the results are compared with CXF spectra in aqueous solution and in methylene chloride. The results indicate that the amide N-H bond is trans to the C=O bond of CXF both in solution and in the enzyme ternary complex. The C=O stretch and N-H bending modes of the amide of CXF shift -16 and -9 cm-1, respectively, in the enzyme ternary complex relative to that in aqueous solution and -48 and 36 cm-1, respectively, relative to that in methylene chloride. Ab initio normal mode calculations on various model systems of CXF show that the observed frequency changes of the C=O stretch mode have contributions from the frequency changes induced by the environmental changes near both the local C=O bond and the remote N-H bond. The same is true for the observed N-H bending frequency change. Our calculations also show that the environmentally induced frequency changes are additive so that it is possible to determine the C=O stretch (or N-H bending) frequency change that is due to the local interaction change near the C=O (or N-H) bond from the observed frequency changes. On the basis of these results and the empirical relationship between the C=O stretch frequency shift and the interaction enthalpy change on the C=O bond developed here, it is found that the C=O group of CXF in the enzyme/NADH/CXF complex binds with a favorable interaction enthalpy of approximately 5.5 kcal/mol relative to water. Similar analysis suggests that the N-H moiety of CXF is destabilized in the ternary complex by about 1.5 kcal/mol relative to water but is stabilized by about 1.5 kcal/mol relative to a hydrophobic environment. The analysis describes quantitatively the binding of the C=O of CXF with the catalytic zinc and the hydroxyl group of Ser-48 and the interaction of the N-H with the benzene ring of Phe-93 of the enzyme.

The controlled study presented here was designed to examine the influence of the degree of structure of a coping-orientated therapy, the changes between the beginning and the end of therapy concerning satisfaction with therapy, psychopathology, attitude towards medication, coping behaviour and social variables, the effects on the days of hospitalisation within one year after the end of treatment and the relevant variables to predict the outcome twelve months later. It could be shown that patients in a relatively unstructured supportive group therapy wished to discuss topics that are very similar to those of coping-orientated therapy, that coping-orientated therapy results especially in an increase in knowledge about illness and treatment and that the therapeutic outcome after a one-year period can best be predicted by the degree of active coping strategies, social adaptation, trust in medication and cognitive variables.

Human alcohol dehydrogenase (HsADH) comprises class I (alpha, beta, and gamma), class II (pi), and class IV (sigma) enzymes. Selective inhibitors of the enzymes could be used to prevent the metabolism of alcohols that form toxic products. Formamides are unreactive analogues of aldehydes and bind to the enzyme-NADH complex [Ramaswamy, S.; Scholze, M.; Plapp, B. V. Biochemistry 1997, 36, 3522-3527]. They are uncompetitive inhibitors against varied concentrations of alcohol, and this makes them effective even with saturating concentrations of alcohols. Molecular modeling led to the design and synthesis of a series of cyclic, linear, and disubstituted formamides. Evaluation of 23 compounds provided structure-function information and selective inhibitors for the enzymes, which have overlapping but differing substrate specificities. Monosubstituted formamides are good inhibitors of class I and II enzymes, and disubstituted formamides are selective for the alpha enzyme. Selective inhibitors, with Ki values at pH 7 and 25 degrees C of 0.33-0.74 microM, include N-cyclopentyl-N-cyclobutylformamide for HsADH alpha, N-benzylformamide for HsADH beta1, N-1-methylheptylformamide for HsADH gamma2, and N-heptylformamide for HsADH sigma and HsADH beta1.

The species of the family Goodeidae have evolved reproductive strategies involving intraovarian gestation, early evacuation of nearly yolk-exhausted embryos from the ovigerous tissue into the ovarian cavity, placental matrotrophy during intraluminal gestation, and the birth of highly developed fry. The inner ovarian lining becomes hypervascularized during gestational periods and functions as the maternal component of the placental association. Embryotrophic liquid is secreted by the inner ovarian epithelium into the ovarian cavity. Comparative electrophoretic analyses of embryotrophe and maternal blood serum provide evidence for the transfer of maternal serum proteins into the embryotrophe. Trophotaeniae, proctodaeal processes of the embryos, provide a surface for nutrient absorption. Endocytic activity was demonstrated by ingestion of unspecific tracer proteins in various species. Moreover, the trophotaenial absorptive cells (TACs) in Ameca splendens ingest various proteins or random copolymers conjugated to colloidal gold as well as radioiodinated proteins in a way that satisfies the criteria of receptor-mediated endocytosis. Several aminopeptidases (APs) on the surface of TACs were identified as protein binding sites as evidenced by inhibition of binding and uptake of marker proteins in the presence of AP substrates or AP inhibitors. Morphological adaptations of the embryonic circulatory system pertaining to nutrient and gas exchange were characterized. The embryonic epidermis comprises two layers of squamous cells closely underlain by a dense capillary net. Efficient gas exchange is facilitated by a thin embryotrophe-blood barrier of both the embryonic skin and the intraovarian lining.

The Popeye domain containing (Popdc) gene family displays preferential expression in skeletal muscle and heart. Only recently a significant gain in the understanding of the function of Popdc genes in the heart has been obtained. The Popdc genes encode membrane proteins harboring an evolutionary conserved Popeye domain, which functions as a binding domain for cyclic adenosine monophosphate (cAMP). Popdc proteins interact with the two-pore channel TREK-1 and enhance its current. This protein interaction is modulated by cAMP. Null mutations of members of the Popdc gene family in zebrafish and mouse are associated with severe cardiac arrhythmia phenotypes. While in zebrafish an atrioventricular block was prevalent, in mouse a stress-induced sinus bradycardia was observed, which was due to the presence of sinus pauses. Moreover, the phenotype develops in an age-dependent manner, being absent in the young animal and becoming increasingly severe, as the animals grow older. This phenotype is reminiscent of the sick sinus syndrome (SSS), which affects mostly the elderly and is characterized by the poor ability of the cardiac pacemaker to adapt the heart rate to the physiological demand. While being a prevalent disease, which is responsible for a large fraction of pacemaker implantations in Western countries, SSS is poorly understood at the molecular level. It is therefore expected that the study of the molecular basis of the stress-induced bradycardia in Popdc mice will shed new light on the etiology of pacemaker disease.

The substrate analogue L-aspartate beta-semialdehyde (L-ASA) has been identified as a mechanism-based inactivator of L-aspartase from Escherichia coli. The enzyme catalyzes the deamination of L-ASA to yield fumaric acid semialdehyde (FAA) and NH4+, with the product FAA partitioning between subsequent release or irreversible enzyme inactivation. Complete protection against L-ASA inactivation is observed in the presence of the product fumarate and a divalent metal ion. However, protection against inactivation by the product FAA also requires the presence of an enzyme activator. In addition to functioning as a mechanism-based inactivator, L-ASA has also been shown to serve as an activator of L-aspartase. The mechanism of inactivation by FAA involves the attack of an active site nucleophilic at the alpha-carbon of FAA to yield a stable Michael type enzyme adduct. Subsequent formation of a hydrazone upon treatment of the enzyme adduct with 2,4-dinitrophenylhydrazine confirms the presence of the unreacted aldehydic group of FAA. Examination of a group of product analogues with different substituents has demonstrated a correlation between the electron-withdrawing ability of these functional groups and the rate of inactivation of L-aspartase.

The potential importance of several functional amino acids in the activity of L-aspartase from Escherichia coli has been examined by site-directed mutagenesis. Amino acids whose importance in enzyme activity was suggested by chemical modification and pH dependence studies were chosen as candidates for investigation. The selection of the particular amino acid targets was guided by homology comparisons among the other sequenced bacterial L-aspartases and by the broader comparison among the fumarase-aspartase enzyme family. Substitution of the most highly conserved cysteine with either serine or alanine, or the most highly conserved histidine with leucine, had no significant effect on the activity of L-aspartase or on the sensitivity of these mutated L-aspartases to cysteine and histidine specific modifying reagents. However, alteration of each of the two conserved lysines to arginine did cause dramatic changes in the catalytic properties of the enzyme. Modification of lysine 54 results in the complete loss of enzyme activity. However, this activity loss appears to be related to changes in the subunit association properties of the arginine 54 mutant. Lysine 326 appears to be involved in substrate binding. Modification of this residue causes a 5-fold increase in the Km for aspartic acid, a drastic decrease in kcat/Km, and a change in the divalent metal ion requirements of the enzyme.

YBa 2 Cu 3 O 7 films have been grown epitaxially on biaxially textured Ni substrates and on single crystalline Ni films by laser ablation. The configuration of the samples was YBa 2 Cu 3 O 7 /yttrium-stabilized zirconia/CeO 2 /Ni in both cases. The YBa 2 Cu 3 O 7 coated conductor sample shows due to the grain boundary network (GBN) a reduced J c value of 0.3 MA/cm 2 ( T =77 K and B =0 T) compared with the J c corresponding to the sample on the single crystalline Ni film of 1.3 MA/cm 2 ( T =77 K and B =0 T). Texture and electron backscattering diffraction measurements of the samples were performed before and after the growth; this allowed a quantitative evaluation of the average misorientation of the GBN, which determines the critical current density.

Teleosts do not possess uteri and ovulation occurs into the ovarian lumen. Hence, osteichthyan viviparity is characterized by intrafollicular gestation or intraluminal gestation (within the ovarian cavity), and thus, the ovary functions both in egg production and maintenance of the developing embryos. Because there is no attachment between embryonic and maternal tissues, maternal-embryonic exchange of gas, electrolytes, nutrients, metabolic waste, and possibly effector molecules and immunoglobulins is mediated by embryotrophe. In several instances it has been demonstrated that embryotrophic proteins originate from maternal serum rather than from elaboration within ovarian tissues. In intraluminal gestation, the ovarian wall becomes hypervascularized and the inner ovarian epithelium lining the ovarian cavity secretes embryotrophe. Trophotaeniae provide extraembryonic endodermal exchange surfaces in many species undergoing intraluminal gestation. There is a strong indication of a trophotaenial membrane binding system for proteins containing appreciable amounts of lysine. In intrafollicular gestation, yolk, pericardial and coelomic sacs play a major role in physiologic exchange. In most instances, intrafollicular gestation is associated with extraembryonic fine structural modifications consistent with respiratory and osmoregulatory functions. The postulated role in nutrient transfer requires experimental evidence. In matrotrophic viviparous teleosts, the gut may provide an effective absorptive surface. On the other hand, the skin, and perhaps the gills and buccopharyngeal tissue, serve as respiratory surfaces. As yet, no experimental evidence suggests exchange of metabolites at these sites. © 1993 Wiley-Liss, Inc.