Hit-to-lead studies on benzimidazole inhibitors of ITK: discovery of a novel class of kinase inhibitors

Benzimidazole 1 was identified as a selective inhibitor of ITK by high throughput screening. Hit-to-lead studies defined the SAR at all three substituents. Reversing the amide linkage at C6 led to 16, with a fivefold improvement of potency. This enhancement is rationalized by the conformational preference of the substituent. A model for the binding of the benzimidazoles to the ATP-binding site of ITK is proposed.     

Livestock husbandry as a tool for carnivore conservation in Africa’s community rangelands: a case–control study

Conflict between people and wildlife is a major issue in both wildlife conservation and rural development. In African rangelands, species such as African wild dogs (Lycaon pictus), cheetahs (Acinonyx jubatus), lions (Panthera leo), leopards (Panthera pardus), and spotted hyaenas (Crocuta crocuta) may kill livestock and are therefore themselves killed by local pastoralists. Such conflict has led to the extirpation of these species from many areas, and also impacts the livelihoods of local livestock farmers. To investigate the possibilities for coexistence of people, livestock, and large predators in community rangelands, we measured the effectiveness of traditional livestock husbandry in reducing depredation by wild carnivores, using a case–control approach. Different measures were effective against different predator species but, overall, the risk of predator attack by day was lowest for small herds, accompanied by herd dogs as well as human herders, grazing in open habitat. By night, the risk of attack was lowest for herds held in enclosures (‘bomas’) with dense walls, pierced by few gates, where both men and domestic dogs were present. Unexpectedly, the presence of scarecrows increased the risks of attack on bomas. Our findings suggest that improvements to livestock husbandry can contribute to the conservation and recovery of large carnivores in community rangelands, although other measures such as prey conservation and control of domestic dog diseases are also likely to be necessary for some species.     

Testicular Sclerosing Sertoli Cell Tumor: A Case Report and Review of the Literature

Sertoli cell tumors are very rare testicular tumors, representing 0.4% to 1.5% of all testicular malignancies. They are subclassified as classic, large-cell calcifying, and sclerosing Sertoli cell tumors (SSCT) based on distinct clinical features. Only 42 cases of SSCTs have been reported in the literature. We present a case of a 23-year-old man diagnosed with SSCT.Key words: Testicular neoplasm, Sertoli cell tumor, Sclerosing Sertoli cell tumorA 23-year-old man was referred to the Cleveland Clinic Department of Urology (Cleveland, OH) for an incidentally detected right testicular mass. The mass was identified during a work-up for transient left testicular discomfort. His only notable medical history was nephrolithiasis. There was no personal or family history of testicular cancer or cryptorchidism. On physical examination, he was a well-nourished, well-masculinized young man without gynecomastia. Testicular examination revealed normal volume and consistency bilaterally without other relevant findings. Testicular ultrasonography demonstrated an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle with peripheral flow on color Doppler (Figure 1).Open in a separate windowFigure 1Testicular ultrasound demonstrating an 8 mm × 6 mm × 6 mm hypoechoic, solid mass in the posterior right testicle (blue arrows).The remainder of the ultrasound examination yielded normal results. Lactic dehydrogenase, B-human chorionic gonadotropin, and α-fetoprotein levels were all within the normal range. After a thorough review of the options, the patient was then taken to the operating room for inguinal exploration. Intraoperative ultrasound confirmed a superficial 8-mm hypoechoic testis lesion. A whiteyellow, well-demarcated nodule was widely excised and a frozen section was sent to pathology for examination. The frozen section examination revealed the lesion to be a neoplasm with differential diagnosis including sclerosing Sertoli cell tumor (SSCT), adenomatoid tumor, and a variant of Leydig cell tumor. Because the final diagnosis could not be determined from frozen section, the decision was made to perform a right radical orchiectomy. Pathologic examination revealed a grossly unifocal, well-circumscribed, white, firm mass of 0.8 cm. Microscopically the lesion was composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Although the lesion was somewhat well circumscribed, entrapped seminiferous tubules with Sertoli-only cells were present within the tumor (Figure 2). Tumor cells had pale or eosinophilic cytoplasm with small and dark nuclei with inconspicuous nucleoli. The tumor was confined to the testis and margins were negative. A diagnosis of SSCT was reached, supported by positive immunostain results for steroidogenic factor 1, focal inhibin, and calretinin expression, and negative stain results for cytokeratin AE1/AE3 and epithelial membrane antigen in the tumor (Figure 3). The postoperative course was unremarkable. Computed tomography scan of the abdomen and pelvis and chest radiograph were negative for metastatic disease.Open in a separate windowFigure 2Low-power examination revealing a well-circumscribed tumor composed of solid and hollow tubules and occasional anastomosing cords distributed within the hypocellular, densely collagenous stroma. Hematoxylin and eosin stain, original magnification ×40. (B) High-power examination. Note entrapped seminiferous tubules lacking spermatogenesis. Hematoxylin and eosin stain, original magnification ×100.Open in a separate windowFigure 3Nuclear expression of steroidogenic factor 1 in the tumor as well as benign Sertoli cells in entrapped seminiferous tubules (original magnification ×200). (B) Focal calretinin expression in the tumor (inhibin had a similar staining pattern; original magnification ×100).     

Nonoverlapping functions of the Polycomb group Cbx family of proteins in embryonic stem cells

Polycomb group proteins are essential regulators of cell fate decisions during embryogenesis. In mammals, at least five different Cbx proteins (Cbx2, Cbx4, Cbx6, Cbx7, and Cbx8) are known to associate with the core Polycomb repressive complex 1 (PRC1). Here we show that pluripotency and differentiation of mouse embryonic stem cells (ESCs) is regulated by different Cbx-associated PRC1 complexes with unique functions. Maintenance of pluripotency primarily depends on Cbx7, while lineage commitment is orchestrated by Cbx2 and Cbx4. At the molecular level, we have uncovered a Polycomb autoregulatory loop in which Cbx7 represses the expression of prodifferentiation Cbx proteins, thereby maintaining the pluripotent state. We additionally show that the occupancy of Cbx7 on promoters is completely dependent on PRC2 activity but only partially dependent on a functional PRC1 complex. Thus, Cbx proteins confer distinct target selectivity to the PRC1 complex, achieving a balance between the self-renewal and the differentiation of ESCs.     

Adverse events among HIV/MDR-TB co-infected patients receiving antiretroviral and second line anti-TB treatment in Mumbai, India

Background

Significant adverse events (AE) have been reported in patients receiving medications for multidrug- and extensively-drug-resistant tuberculosis (MDR-TB & XDR-TB). However, there is little prospective data on AE in MDR- or XDR-TB/HIV co-infected patients on antituberculosis and antiretroviral therapy (ART) in programmatic settings.

Methods

Médecins Sans Frontières (MSF) is supporting a community-based treatment program for drug-resistant tuberculosis in HIV-infected patients in a slum setting in Mumbai, India since 2007. Patients are being treated for both diseases and the management of AE is done on an outpatient basis whenever possible. Prospective data were analysed to determine the occurrence and nature of AE.

Results

Between May 2007 and September 2011, 67 HIV/MDR-TB co-infected patients were being treated with anti-TB treatment and ART; 43.3% were female, median age was 35.5 years (Interquartile Range: 30.5–42) and the median duration of anti-TB treatment was 10 months (range 0.5–30). Overall, AE were common in this cohort: 71%, 63% and 40% of patients experienced one or more mild, moderate or severe AE, respectively. However, they were rarely life-threatening or debilitating. AE occurring most frequently included gastrointestinal symptoms (45% of patients), peripheral neuropathy (38%), hypothyroidism (32%), psychiatric symptoms (29%) and hypokalaemia (23%). Eleven patients were hospitalized for AE and one or more suspect drugs had to be permanently discontinued in 27 (40%). No AE led to indefinite suspension of an entire MDR-TB or ART regimen.

Conclusions

AE occurred frequently in this Mumbai HIV/MDR-TB cohort but not more frequently than in non-HIV patients on similar anti-TB treatment. Most AE can be successfully managed on an outpatient basis through a community-based treatment program, even in a resource-limited setting. Concerns about severe AE in the management of co-infected patients are justified, however, they should not cause delays in the urgently needed rapid scale-up of antiretroviral therapy and second-line anti-TB treatment.     

ERK1/2 and p38 MAPKs Are Complementarily Involved in Estradiol 17?-d-Glucuronide-Induced Cholestasis: Crosstalk with cPKC and PI3K

Objective

The endogenous, cholestatic metabolite estradiol 17ß-d-glucuronide (E₂17G) induces endocytic internalization of the canalicular transporters relevant to bile formation, Bsep and Mrp2. We evaluated here whether MAPKs are involved in this effect.

Design

ERK1/2, JNK1/2, and p38 MAPK activation was assessed by the increase in their phosphorylation status. Hepatocanalicular function was evaluated in isolated rat hepatocyte couplets (IRHCs) by quantifying the apical secretion of fluorescent Bsep and Mrp2 substrates, and in isolated, perfused rat livers (IPRLs), using taurocholate and 2,4-dinitrophenyl-S-glutathione, respectively. Protein kinase participation in E₂17G-induced secretory failure was assessed by co-administering selective inhibitors. Internalization of Bsep/Mrp2 was assessed by confocal microscopy and image analysis.

Results

E₂17G activated all kinds of MAPKs. The PI3K inhibitor wortmannin prevented ERK1/2 activation, whereas the cPKC inhibitor Gö6976 prevented p38 activation, suggesting that ERK1/2 and p38 are downstream of PI3K and cPKC, respectively. The p38 inhibitor SB203580 and the ERK1/2 inhibitor PD98059, but not the JNK1/2 inhibitor SP600125, partially prevented E₂17G-induced changes in transporter activity and localization in IRHCs. p38 and ERK1/2 co-inhibition resulted in additive protection, suggesting complementary involvement of these MAPKs. In IPRLs, E₂17G induced endocytosis of canalicular transporters and a rapid and sustained decrease in bile flow and biliary excretion of Bsep/Mrp2 substrates. p38 inhibition prevented this initial decay, and the internalization of Bsep/Mrp2. Contrarily, ERK1/2 inhibition accelerated the recovery of biliary secretion and the canalicular reinsertion of Bsep/Mrp2.

Conclusions

cPKC/p38 MAPK and PI3K/ERK1/2 signalling pathways participate complementarily in E₂17G-induced cholestasis, through internalization and sustained intracellular retention of canalicular transporters, respectively.     

Biology and ecology of <Emphasis Type="Italic">Appendiseta robiniae</Emphasis> (Hemiptera: Aphidoidea) — an alien species in Europe

Elements of the biology and ecology of A. robiniae were studied in Poland during the period of 2008–2009. The term of spring hatching, number of generations per season, developmental time of particular generations, lifespan of specimens and fecundity of particular generations were all defined. The dynamics of the species’ occurrence on its host plant were also determined. The two-year study showed a maximum of 11 aphid generations can develop on Robinia pseudoacacia. Females of the second and third generations were found to be the most fertile. These findings indicate that the species has adapted well in Poland and its biological predispositions have enabled it to rapidly increase in population.     

Synthesis, antiplatelet activity and comparative molecular field analysis of substituted 2-amino-4H-pyrido[1,2-a]pyrimidin-4-ones, their congeners and isosteric analogues

2-(1-Piperazinyl)-4H-pyrido[1,2-a]pyrimidin-4-one (5a) is a recently described in vitro inhibitor of human platelet aggregation which specifically inhibits the activity of high affinity cAMP phosphodiesterase. A number of substitution derivatives, isosteres, and analogues of 5a were now synthesized and tested in vitro for their inhibitory activity on human platelet aggregation induced in platelet-rich plasma by ADP, collagen, or the Ca2+ ionophore A23187. Among the most effective compounds, the 6-methyl, 8-methyl and 6,8-dimethyl derivatives of 5a resulted nearly as active as the lead when platelet aggregation was induced by ADP or A23187, but less active when collagen was the inducer. On the basis of present results and those previously obtained by us in this and 2-aminochromone structural fields, we have developed a statistically significant 3-D QSAR model, using comparative molecular field analysis (CoMFA), describing the variation of the antiplatelet activity in terms of molecular steric and electrostatic potential changes.     

Leaf age as a factor in anatomical and physiological acclimative responses of Taxus baccata L. needles to contrasting irradiance environments

To evaluate the acclimative ability of current-year and previous-year needles of a shade tolerant conifer Taxus baccata L. to contrasting irradiance conditions, seedlings were raised under 27% solar irradiance and at 3 years of age they were transferred to an experimental garden and grown for one season under full irradiance (HL), 18% irradiance (ML) or 5% irradiance (LL). Whereas previous year needles did not change anatomically, current year needles in HL were thicker and had a thicker palisade and spongy mesophyll, and greater leaf mass per area than ML or LL needles. LL needles had greater nitrogen concentration than HL needles irrespective of age but only previous year LL needles also had an increased N per area content, thanks to their lack of reduction in LMA. Adjustment of chlorophyll and carotenoid content occurred in both needle age classes with LL and ML needles having much higher concentrations but, in current year needles, only slightly higher per area content than HL needles. Chlorophyll a/b ratio was not affected by age or irradiance. These modifications had no significant effect on photosynthetic capacities, which did not significantly differ between the age classes in HL or LL treatment and between treatments. On the other hand, high growth irradiance resulted in a greater photochemical yield, photochemical quenching, apparent electron transport rate and inducible non-photochemical quenching in needles formed in the current season. In previous year needles, however, only inducible NPQ was enhanced by high irradiance with other parameters remaining identical among treatments. To test sensitivity to photoinhibition, at the end of the summer plants from the three irradiance levels were transferred to a HL situation and F _v/F _M was determined over the following 18 days. Sensitivity to photoinhibition was negatively related to growth irradiance and previous year needles were less photoinhibited than current year needles. Thus, differences in acclimation ability between needle age classes were most pronounced at the level of anatomy and light reactions of photosynthesis, both of which showed almost no plasticity in previous year needles but were considerably modified by irradiance in current year needles.