Combined Application of Pan-AKT Inhibitor MK-2206 and BCL-2 Antagonist Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia.

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 on B-ALL cell lines and primary samples and investigate potential synergistic effects with BCL-2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK-2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK-2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK-2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4;11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK-2206. Functional assessment of BCL-2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK-2206 and venetoclax incubation. In summary, we demonstrate that the pan-AKT inhibitor MK-2206 potently blocks B-ALL cell proliferation and for the first time characterize the synergistic effect of combined MK-2206 and venetoclax treatment in B-ALL.

Distribution and phylogeny of Brachyspira spp. in human intestinal spirochetosis revealed by FISH and 16S rRNA-gene analysis.

During six years as German National Consultant Laboratory for Spirochetes we investigated 149 intestinal biopsies from 91 patients, which were histopathologically diagnosed with human intestinal spirochetosis (HIS), using fluorescence in situ hybridization (FISH) combined with 16S rRNA gene PCR and sequencing. Aim of this study was to complement histopathological findings with FISH and PCR for definite diagnosis and species identification of the causative pathogens. HIS is characterized by colonization of the colonic mucosa of the human distal intestinal tract by Brachyspira spp. Microbiological diagnosis of HIS is not performed, because of the fastidious nature and slow growth of Brachyspira spp. in culture. In clinical practice, diagnosis of HIS relies solely on histopathology without differentiation of the spirochetes. We used a previously described FISH probe to detect and identify Brachyspira spp. in histological gut biopsies. FISH allowed rapid visualization and identification of Brachyspira spp. in 77 patients. In most cases, the bright FISH signal already allowed rapid localization of Brachyspira spp. at 400× magnification. By sequencing, 53 cases could be assigned to the B. aalborgi lineage including "B. ibaraki" and "B. hominis", and 23 cases to B. pilosicoli. One case showed mixed colonization. The cases reported here reaffirm all major HIS Brachyspira spp. clusters already described. However, the phylogenetic diversity seems to be even greater than previously reported. In 14 cases, we could not confirm HIS by either FISH or PCR, but found colonization of the epithelium by rods and cocci, indicating misdiagnosis by histopathology. FISH in combination with molecular identification by 16S rRNA gene sequencing has proved to be a valuable addition to histopathology. It provides definite diagnosis of HIS and allows insights into phylogeny and distribution of Brachyspira spp. HIS should be considered as a differential diagnosis in diarrhea of unknown origin, particularly in patients from risk groups (e.g. patients with colonic adenomas, inflammatory polyps, inflammatory bowel disease or HIV infection and in men who have sex with men).

Fluorescence in situ hybridization for the identification of Treponema pallidum in tissue sections.

Syphilis is often called the great imitator because of its frequent atypical clinical manifestations that make the disease difficult to recognize. Because Treponema pallidum subsp. pallidum, the infectious agent of syphilis, is yet uncultivated in vitro, diagnosis is usually made using serology; however, in cases where serology is inconclusive or in patients with immunosuppression where these tests may be difficult to interpret, the availability of a molecular tool for direct diagnosis may be of pivotal importance. Here we present a fluorescence in situ hybridization (FISH) assay that simultaneously identifies and analyzes spatial distribution of T. pallidum in histological tissue sections. For this assay the species-specific FISH probe TPALL targeting the 16S rRNA of T. pallidum was designed in silico and evaluated using T. pallidum infected rabbit testicular tissue and a panel of non-syphilis spirochetes as positive and negative controls, respectively, before application to samples from four syphilis-patients. In a HIV positive patient, FISH showed the presence of T. pallidum in inguinal lymph node tissue. In a patient not suspected to suffer from syphilis but underwent surgery for phimosis, numerous T. pallidum cells were found in preputial tissue. In two cases with oral involvement, FISH was able to differentiate T. pallidum from oral treponemes and showed infection of the oral mucosa and tonsils, respectively. The TPALL FISH probe is now readily available for in situ identification of T. pallidum in selected clinical samples as well as T. pallidum research applications and animal models.

Neuropeptidergic input pathways to the circadian pacemaker center of the Madeira cockroach analysed with an improved injection technique.

Light entrainment pathways synchronize the circadian clock of almost all species of the animal and plant kingdom to the daily light dark cycle. In the Madeira cockroach Rhyparobia (Leucophaea) maderae, the circadian clock is located in the accessory medulla of the brain's optic lobes. The clock has abundant neuropeptides with unknown functions. Previous studies suggested that myoinhibitory peptides (MIPs), orcokinins (ORCs), and allatotropin (AT) take part in light input pathways to the circadian clock. As the sequences of AT and ORCs of R. maderae have not yet been determined, with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the respective Rhyparobia peptides were characterized. To search for light-like phase-shifting inputs to the circadian clock, Rhyparobia-MIP-1, Rhyparobia-AT, and Rhyparobia-ORC were injected at different circadian times, combined with locomotor activity assays. An improved, less invasive injection method was developed that allowed for the analysis of peptide effects within <2 weeks after injection. Rhyparobia-MIP-1 and Rhyparobia-AT injections resulted in dose-dependent monophasic phase response curves with maximum delays at the beginning of the subjective night, similar to light-dependent phase delays. In contrast to Manduca sexta-AT, Rhyparobia-AT did not phase advance locomotor activity rhythms. Only injections of Rhyparobia-ORCs resulted in a biphasic light-like phase response curve. Thus, it is hypothesized that Rhyparobia-MIP-1 and -AT are candidates for relaying light-dependent delays and/or non-photic inputs to the clock, whereas Rhyparobia-ORCs might be part of the light-entrainment pathways relaying phase delays and advances to the circadian clock of the Madeira cockroach.

The lab oddity prevails: discovery of pan-CDK inhibitor (R)-S-cyclopropyl-S-(4-{[4-{[(1R,2R)-2-hydroxy-1-methylpropyl]oxy}-5-(trifluoromethyl)pyrimidin-2-yl]amino}phenyl)sulfoximide (BAY 1000394) for the treatment of cancer.

Lead optimization of a high-throughput screening hit led to the rapid identification of aminopyrimidine ZK 304709, a multitargeted CDK and VEGF-R inhibitor that displayed a promising preclinical profile. Nevertheless, ZK 304709 failed in phase I studies due to dose-limited absorption and high inter-patient variability, which was attributed to limited aqueous solubility and off-target activity against carbonic anhydrases. Further lead optimization efforts to address the off-target activity profile finally resulted in the introduction of a sulfoximine group, which is still a rather unusual approach in medicinal chemistry. However, the sulfoximine series of compounds quickly revealed very interesting properties, culminating in the identification of the nanomolar pan-CDK inhibitor BAY 1000394, which is currently being investigated in phase I clinical trials.

Imaging properties of small-pixel spectroscopic x-ray detectors based on cadmium telluride sensors.

Spectroscopic x-ray imaging by means of photon counting detectors has received growing interest during the past years. Critical to the image quality of such devices is their pixel pitch and the sensor material employed. This paper describes the imaging properties of Medipix2 MXR multi-chip assemblies bump bonded to 1 mm thick CdTe sensors. Two systems were investigated with pixel pitches of 110 and 165 µm, which are in the order of the mean free path lengths of the characteristic x-rays produced in their sensors. Peak widths were found to be almost constant across the energy range of 10 to 60 keV, with values of 2.3 and 2.2 keV (FWHM) for the two pixel pitches. The average number of pixels responding to a single incoming photon are about 1.85 and 1.45 at 60 keV, amounting to detective quantum efficiencies of 0.77 and 0.84 at a spatial frequency of zero. Energy selective CT acquisitions are presented, and the two pixel pitches' abilities to discriminate between iodine and gadolinium contrast agents are examined. It is shown that the choice of the pixel pitch translates into a minimum contrast agent concentration for which material discrimination is still possible. We finally investigate saturation effects at high x-ray fluxes and conclude with the finding that higher maximum count rates come at the cost of a reduced energy resolution.

Histone H3 lysine 36 methylation targets the Isw1b remodeling complex to chromatin.

Histone H3 lysine 36 methylation is a ubiquitous hallmark of productive transcription elongation. Despite the prevalence of this histone posttranslational modification, however, the downstream functions triggered by this mark are not well understood. In this study, we showed that H3K36 methylation promoted the chromatin interaction of the Isw1b chromatin-remodeling complex in Saccharomyces cerevisiae. Similar to H3K36 methylation, Isw1b was found at the mid- and 3' regions of transcribed genes genome wide, and its presence at active genes was dependent on H3K36 methylation and the PWWP domain of the Isw1b subunit, Ioc4. Moreover, purified Isw1b preferentially interacted with recombinant nucleosomes that were methylated at lysine 36, and this interaction also required the Ioc4 PWWP domain. While H3K36 methylation has been shown to regulate the binding of numerous factors, this is the first time that it has been shown to facilitate targeting of a chromatin-remodeling complex.

Myoinhibitory peptides in the brain of the cockroach Leucophaea maderae and colocalization with pigment-dispersing factor in circadian pacemaker cells.

Myoinhibitory peptides (MIPs) are a family of insect W(X(6))Wamides with inhibitory effects on visceral muscles and juvenile hormone synthesis. Although MIPs are widely distributed within the nervous system, a detailed analysis of their distribution and function in insect brains is still missing. We analyzed the distribution of MIPs in the brain of the cockroach Leucophaea maderae. We focused on the accessory medulla (AMe), a small neuropil near the medulla that acts as the master circadian clock. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and Nano-LC electrospray ionization (ESI) mass spectrometry revealed five Lem-MIPs in preparations of the AMe and corpora cardiaca. The complete sequences of two of these peptides were identified. Immunocytochemistry revealed wide distribution of MIP-related peptides in the cockroach brain. The superior median protocerebrum, parts of the central complex, and the tritocerebrum showed particularly dense immunostaining. In contrast, only a few local interneurons were stained in the antennal lobe and a few extrinsic neurons in the mushroom body, including a giant neuron innervating the calyces. The noduli of the AMe showed dense immunostaining, and neurons in all AMe cell groups except the anterior neurons were labeled. Pigment-dispersing factor- (PDF) and MIP immunostaining was colocalized in two neurons of the AMe. No colocalization of MIP- and PDF immunostaining was detected in the anterior optic commissure, but two small PDF-immunoreactive commissural fibers near the posterior optic commissure showed colocalized MIP immunostaining. The results suggest that several MIPs participate in different functional circuits of the circadian system and are involved in multiple brain circuits of the Madeira cockroach.

YEATS domain proteins: a diverse family with many links to chromatin modification and transcription.

Chromatin modifications play crucial roles in various biological processes. An increasing number of conserved protein domains, often found in multisubunit protein complexes, are involved in establishing and recognizing different chromatin modifications. The YEATS domain is one of these domains, and its role in chromatin modifications and transcription is just beginning to be appreciated. The YEATS domain family of proteins, conserved from yeast to human, contains over 100 members in more than 70 eukaryotic species. Yaf9, Taf14, and Sas5 are the only YEATS domain proteins in Saccharomyces cerevisiae. Human YEATS domain family members, such as GAS41, ENL, and AF9, have a strong link to cancer. GAS41 is amplified in glioblastomas and astrocytomas; ENL and AF9 are among the most frequent translocation partners of the mixed lineage leukemia (MLL) gene. This review will focus on the best characterized YEATS proteins, discuss their diverse roles, and reflect potential functions of the YEATS domain.

Acute psychological stress alerts the adaptive immune response: stress-induced mobilization of effector T cells.

Influences of psychological stress on the acquired immune system have not consequently been investigated. We found acute psychological stress to cause an increase in CD56+ and CCR5+ effector T cells in the peripheral blood of healthy human subjects (N=22), while skin-homing CLA+ T cells decreased. At the same time, we observed a stress-induced decrease in CD45RA+/CCR7+ naive and CD45RA-/CCR7+ central memory T cells, while CD45RA-/CCR7- effector memory and CD45RA+/CCR7- terminally differentiated T cells increased. This T cell redistribution translated into an increase in T cells expressing perforin/granzyme B and in Epstein-Barr virus-specific, cytomegalovirus-specific and influenza virus-specific CD8+ T cells. Thus, acute stress seems to promote the retention of less mature T cells within lymphoid tissue or skin while effector-type T cells are mobilized into the blood in order to be able to rapidly migrate into peripheral tissues.