Identification of Novel Mt-Guab2 Inhibitor Series Active against M. tuberculosis

Tuberculosis (TB) remains a leading cause of mortality worldwide. With the emergence of multidrug resistant TB, extensively drug resistant TB and HIV-associated TB it is imperative that new drug targets be identified. The potential of Mycobacterium tuberculosis inosine monophosphate dehydrogenase (IMPDH) as a novel drug target was explored in the present study. IMPDH exclusively catalyzes the conversion of inosine monophosphate (IMP) to xanthosine monophosphate (XMP) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD+). Although the enzyme is a dehydrogenase, the enzyme does not catalyze the reverse reaction i.e. the conversion of XMP to IMP. Unlike other bacteria, M. tuberculosis harbors three IMPDH-like genes, designated as Mt-guaB1, Mt-guaB2 and Mt-guaB3 respectively. Of the three putative IMPDH's, we previously confirmed that Mt-GuaB2 was the only functional ortholog by characterizing the enzyme kinetically. Using an in silico approach based on designed scaffolds, a series of novel classes of inhibitors was identified. The inhibitors possess good activity against M. tuberculosis with MIC values in the range of 0.4 to 11.4 µg mL−1. Among the identified ligands, two inhibitors have nanomolar Kis against the Mt-GuaB2 enzyme

Transformation and tumorigenicity testing of simian cell lines and evaluation of poliovirus replication

The key role of cell cultures in different scientific fields is worldwide recognized, both as in vitro research models alternative to laboratory animals and substrates for biological production. However, many safety concerns rise from the use of animal/human cell lines that may be tumorigenic, leading to potential adverse contaminations in cell-derived biologicals. In order to evaluate the suitability of 13 different cell lines for Poliovirus vaccine production, safety and quality, in vitro/in vivo tumorigenicity and Poliovirus propagation properties were evaluated. Our results revealed that non-human primate cell lines CYNOM-K1, FRhK-4, 4MBr-5 and 4647 are free of tumorigenic features and represent highly susceptible substrates for attenuated Sabin Poliovirus strains. In particular, FRhK-4 and 4647 cell lines are characterized by a higher in vitro replication, resulting indicated for the use in large-scale production field

Cdh11 Acts as a Tumor Suppressor in a Murine Retinoblastoma Model by Facilitating Tumor Cell Death

CDH11 gene copy number and expression are frequently lost in human retinoblastomas and in retinoblastomas arising in TAg-RB mice. To determine the effect of Cdh11 loss in tumorigenesis, we crossed Cdh11 null mice with TAg-RB mice. Loss of Cdh11 had no gross morphological effect on the developing retina of Cdh11 knockout mice, but led to larger retinal volumes in mice crossed with TAg-RB mice (p = 0.01). Mice null for Cdh11 presented with fewer TAg-positive cells at postnatal day 8 (PND8) (p = 0.01) and had fewer multifocal tumors at PND28 (p = 0.016), compared to mice with normal Cdh11 alleles. However, tumor growth was faster in Cdh11-null mice between PND8 and PND84 (p = 0.003). In tumors of Cdh11-null mice, cell death was decreased 5- to 10-fold (p<0.03 for all markers), while proliferation in vivo remained unaffected (p = 0.121). Activated caspase-3 was significantly decreased and β-catenin expression increased in Cdh11 knockdown experiments in vitro. These data suggest that Cdh11 displays tumor suppressor properties in vivo and in vitro in murine retinoblastoma through promotion of cell death

Citral Sensing by TRANSient Receptor Potential Channels in Dorsal Root Ganglion Neurons

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1–3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin

A Screening Pipeline for Antiparasitic Agents Targeting Cryptosporidium Inosine Monophosphate Dehydrogenase

Persistent diarrhea is a leading cause of illness and death among impoverished children, and a growing share of this disease burden can be attributed to the parasite Cryptosporidium. There are no vaccines to prevent Cryptosporidium infection, and the treatment options are limited and unreliable. Critically, no effective treatment exists for children or adults suffering from AIDS. Cryptosporidium presents many technical obstacles for drug discovery; perhaps the most important roadblock is the difficulty of monitoring drug action. Here we have developed a set of methods to accelerate the drug discovery process for cryptosporidiosis. We exploit the opportunities for experimental manipulation in the related parasite Toxoplasma to genetically engineer a Cryptosporidium model. This new model parasite mirrors the metabolism of Cryptosporidium for a particularly promising drug target that supplies the building blocks for DNA and RNA. Drug effectiveness can be assayed through simple fluorescence measurements for many candidates. Using this assay as an initial filter, and adapting other assays to a high throughput format, we identify several novel chemical compounds that exhibit markedly improved anti-cryptosporidial activity and excellent selectivity

Chelators in Iron and Copper Toxicity

Purpose of Review Chelation therapy is used for diseases causing an imbalance of iron levels (for example haemochromatosis and thalassaemia) or copper levels (for example Menkes’ and Wilson’s diseases). Currently, most pharmaceutical chelators are relatively simple but often have side effects. Some have been taken off the market. This review attempts to find theory and knowledge required to design or find better chelators. Recent Findings Recent research attempting to understand the biological mechanisms of protection against iron and copper toxicity is reviewed. Understanding of molecular mechanisms behind normal iron/copper regulation may lead to the design of more sophisticated chelators. The theory of metal ion toxicity explains why some chelators, such as EDTA, which chelate metal ions in a way which exposes the ion to the surrounding environment are shown to be unsuitable except as a means of killing cancer cells. The Lewis theory of acids and bases suggests which amino acids favour the attachment of the hard/intermediate ions Fe2+, Fe3+, Cu2+ and soft ion Cu+. Non-polar amino acids will chelate the ion in a position not in contact with the surrounding cellular environment. The conclusion is that only the soft ion binding cysteine and methionine appear as suitable chelators. Clearly, nature has developed proteins which are less restricted. Recent research on naturally produced chelators such as siderophores and phytochemicals show some promise as pharmaceuticals. Summary Although an understanding of natural mechanisms of Fe/Cu regulation continues to increase, the pharmaceutical chelators for metal overload diseases remain simple non-protein molecules. Natural and synthetic alternatives have been studied but require further research before being accepted

Insight dimensions and cognitive function in psychosis: a longitudinal study

BACKGROUND: It has been reported that lack of insight is significantly associated with cognitive disturbance in schizophrenia. This study examines the longitudinal relationships between insight dimensions and cognitive performance in psychosis. METHODS: Participants were 75 consecutively admitted inpatients with schizophrenia, affective disorder with psychotic symptoms or schizoaffective disorder. Assessments were conducted at two time points during the study: at the time of hospital discharge after an acute psychotic episode and at a follow-up time that occurred more than 6 months after discharge. A multidimensional approach of insight was chosen and three instruments for its assessment were used: the Scale to Assess Unawareness of Mental Disorder (SUMD), three items concerning insight on the Assessment and Documentation in Psychopathology (AMDP) system and the Insight and Treatment Attitudes Questionnaire. The neuropsychological battery included a wide range of tests that assessed global cognitive function, attention, memory, and executive functions. RESULTS: After conducting adequate statistical correction to avoid Type I bias, insight dimensions and cognitive performance were not found to be significantly associated at cross-sectional and longitudinal assessments. In addition, baseline cognitive performance did not explain changes in insight dimensions at follow-up. Similar results were found in the subset of patients with schizophrenia (n = 37). The possibility of a Type II error might have increased due to sample attrition at follow-up. CONCLUSION: These results suggest that lack of insight dimensions and cognitive functioning may be unrelated phenomena in psychosis

Potential neoplastic evolution of Vero cells: in vivo and in vitro characterization

Vero cell lines are extensively employed in viral vaccine manufacturing. Similarly to all established cells, mutations can occur during Vero cells in vitro amplification which can result in adverse features compromising their biological safety. To evaluate the potential neoplastic evolution of these cells, in vitro transformation test, gene expression analysis and karyotyping were compared among low- (127 and 139 passages) and high-passage (passage 194) cell lines, as well as transformed colonies (TCs). In vivo tumorigenicity was also tested to confirm preliminary in vitro data obtained for low passage lines and TCs. Moreover, Vero cells cultivated in foetal bovine serum-free medium and derived from TCs were analysed to investigate the influence of cultivation methods on tumorigenic evolution. Low-passage Vero developed TCs in soft agar, without showing any tumorigenic evolution when inoculated in the animal model. Karyotyping showed a hypo-diploid modal chromosome number and rearrangements with no difference among Vero cell line passages and TCs. These abnormalities were reported also in serum-free cultivated Vero. Gene expression revealed that high-passage Vero cells had several under-expressedand a few over-expressed genes compared to low-passage ones.Gene ontology revealed no significant enrichment of pathways related to oncogenic risk. These findings suggest that in vitro high passage, and not culture conditions, induces Vero transformation correlated to karyotype and gene expression alterations. These data, together with previous investigations reporting tumour induction in high-passage Vero cells, suggest the use of low-passage Vero cells or cell lines other than Vero to increase the safety of vaccine manufacturing

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta