4-(3-Alkyl/benzyl-guanidino)benzenesulfonamides as selective carbonic anhydrase VII inhibitors

The treatment of chronic neuropathic pain remains one of the most challenging of all neurological diseases and very much an art. There exists no consensus for the optimal management of this condition at the moment. Gaining inspiration from recent studies which pointed out the involvement of brain-associated carbonic anhydrase (CA, EC 4.2.1.1) isoform VII in the pathology of various neurodegenerative diseases, which highlighted the relationship between selective inhibition of this isozyme and relieve of neuropathic pain, herein we report the synthesis and CA VII inhibitory activity of novel 4-(3-alkyl/benzyl-guanidino)benzenesulfonamides. Ten benzyl-substituted and five alkyl-substituted 4-guanidinobenzenesulfonamide derivatives were obtained, some of which (7c, 7h, 7m and 7o) exhibited satisfactory selectivity towards CA VII over CA I and II, with K_I-s in the subnanomolar range and good selectivity indexes for inhibiting the target versus the off-target isoforms.     

Improved fermentation processes for NS0 cell lines expressing human antibodies and glutamine synthetase

To meet the increasing requirement for therapeutic antibodies to conduct clinical trials, an enhanced culture medium and fed-batch process was developed for GS-NS0 cell lines. This process was shown to produce high concentrations of monoclonal antibodies for several cell lines expressing different antibodies. Cells were adapted to growth in a glutamine- and serum-free medium containing bovine serum albumin (BSA), cholesterol, and transferrin. A number of amino acids were found to be depleted during cell culture. The concentrations of these amino acids were increased, and further cell culture analyses were performed. This process of cell growth and analysis was repeated over multiple cycles until no depletion was detected. This resulted in an amino acid supplement that was shown to be generic and enhanced antibody productivity up to 5-fold for the three cell lines tested. Transferrin was replaced using tropolone, a lipophilic iron chelator and ferric ammonium citrate. Cell growth was equivalent to that in transferrin-containing medium over the wide ranges tested. A concentrated feed solution, based on the amino acid supplement and the components of the serum- and protein-free supplements, was formulated. Addition of this feed in response to metabolic requirements resulted in a harvest titer a further 2-fold higher than the enhanced culture medium. Harvest antibody titers of up to 600 mg/L were achieved for three cell lines expressing different antibodies, representing an increase of 10-fold over the starting concentrations.     

From sneaker to parental male: change of reproductive traits in the black goby, Gobius niger (Teleostei, Gobiidae)

This study focuses on the consequences of the switch of tactic from parasitic to parental male in the black goby, Gobius niger (Teleostei: Gobiidae), a species showing two alternative male mating tactics. Older and larger males defend nests, court, and perform parental care on eggs, while younger and smaller ones behave as parasites, sneaking into nests while spawning occurs. Males adopting different tactics are known to present differences in primary and secondary sex traits. The social context of sneaker males was manipulated to induce a tactic switch. Sneakers were kept under two different experimental treatments with or without a female, and under exclusion of male-male competition. Males changed tactics, courting females, spawning, and performing parental care. All males showed substantial changes in primary sexual traits, such as a reduction in gonadal development and an increase in the investment in accessory structures. The experimental groups differed in the functionality of gonads and accessory organs and in the development of the secondary sex traits. These results demonstrate that the moment of switching is not genetically fixed in the black goby. Sneaker males are able to quickly reallocate energy in primary and secondary sex traits, in accordance with the adopted tactic. Several aspects of this flexible reproductive pattern resemble the socially controlled sex change found in sequential hermaphrodites.     

A carbapenem-resistant Klebsiella pneumoniae isolate harboring KPC-1 from Italy

Carbapenem-resistant gram-negative pathogens represent an emerging threat to the management of hospital-acquired infections. Although the isolation of carbapenem-resistant enterobacteriaceae remains unusual, the frequency of carbapenemases producing Klebsiella pneumoniae is increasing in different geographic regions: the majority of isolates has been collected in the USA, but recently KPC-producing K. pneumoniae were reported from China, Israel, Greece, France, Norway and Sweden. We report a KPC 1-producing K. pneumoniae isolate from Italy. This datum enlarges the geographical area where the KPC-producing K. pneumoniae strains are diffuse.     

Staphylococcus aureus cell wall structure and dynamics during host-pathogen interaction

Peptidoglycan is the major structural component of the Staphylococcus aureus cell wall, in which it maintains cellular integrity, is the interface with the host, and its synthesis is targeted by some of the most crucial antibiotics developed. Despite this importance, and the wealth of data from in vitro studies, we do not understand the structure and dynamics of peptidoglycan during infection. In this study we have developed methods to harvest bacteria from an active infection in order to purify cell walls for biochemical analysis ex vivo. Isolated ex vivo bacterial cells are smaller than those actively growing in vitro, with thickened cell walls and reduced peptidoglycan crosslinking, similar to that of stationary phase cells. These features suggested a role for specific peptidoglycan homeostatic mechanisms in disease. As S. aureus missing penicillin binding protein 4 (PBP4) has reduced peptidoglycan crosslinking in vitro its role during infection was established. Loss of PBP4 resulted in an increased recovery of S. aureus from the livers of infected mice, which coincided with enhanced fitness within murine and human macrophages. Thicker cell walls correlate with reduced activity of peptidoglycan hydrolases. S. aureus has a family of 4 putative glucosaminidases, that are collectively crucial for growth. Loss of the major enzyme SagB, led to attenuation during murine infection and reduced survival in human macrophages. However, loss of the other three enzymes Atl, SagA and ScaH resulted in clustering dependent attenuation, in a zebrafish embryo, but not a murine, model of infection. A combination of pbp4 and sagB deficiencies resulted in a restoration of parental virulence. Our results, demonstrate the importance of appropriate cell wall structure and dynamics during pathogenesis, providing new insight to the mechanisms of disease.     

Tandemly duplicated Arabidopsis genes that encode polygalacturonase-inhibiting proteins are regulated coordinately by different signal transduction pathways in response to fungal infection

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins that counteract fungal polygalacturonases, which are important virulence factors. Like many other plant defense proteins, PGIPs are encoded by gene families, but the roles of individual genes in these families are poorly understood. Here, we show that in Arabidopsis, two tandemly duplicated PGIP genes are upregulated coordinately in response to Botrytis cinerea infection, but through separate signal transduction pathways. AtPGIP2 expression is mediated by jasmonate and requires COI1 and JAR1, whereas AtPGIP1 expression is upregulated strongly by oligogalacturonides but is unaffected by salicylic acid, jasmonate, or ethylene. Both AtPGIP1 and AtPGIP2 encode functional inhibitors of polygalacturonase from Botrytis, and their overexpression in Arabidopsis significantly reduces Botrytis disease symptoms. Therefore, gene duplication followed by the divergence of promoter regions may result in different modes of regulation of similar defensive proteins, thereby enhancing the likelihood of defense gene activation during pathogen infection.     

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1–42 (Aβ) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aβ boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aβ-induced effects.Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aβ and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites.As a whole, the collected data revealed that, the damaging path induced by Aβ in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.     

Mobile Communication in the Public Mind: Insights from Free Associations Related to Mobile Phone Base Stations

The number of people using mobile phones has dramatically increased. At the same time, many people are unsettled about the potential health effects from the electromagnetic fields generated by mobile phone base stations. Research indicates that the risks associated with base stations are perceived differently by experts, laypeople, and base station opponents. Using a free association method, we analyzed these differences in more detail. In our first study, we found no difference between experts and laypeople but a marked distinction in the associations of opponents as opposed to the first two groups. The prevalence of free associations in a large random sample from the general population was explored via correspondence analysis in the second study. People who assign high risks to mobile communication had different, more negative associations in mind (e.g., “senselessness,” “hazard”) compared to people with low risk-perception (e.g., “mast,” “acceptance”). Our research is in line with the assumption that the affect heuristic guides risk and benefit assessments, and highlights the role of affect in risk perception and communication.     

Exome Sequencing Identifies Rare Deleterious Mutations in DNA Repair Genes FANCC and BLM as Potential Breast Cancer Susceptibility Alleles

Despite intensive efforts using linkage and candidate gene approaches, the genetic etiology for the majority of families with a multi-generational breast cancer predisposition is unknown. In this study, we used whole-exome sequencing of thirty-three individuals from 15 breast cancer families to identify potential predisposing genes. Our analysis identified families with heterozygous, deleterious mutations in the DNA repair genes FANCC and BLM, which are responsible for the autosomal recessive disorders Fanconi Anemia and Bloom syndrome. In total, screening of all exons in these genes in 438 breast cancer families identified three with truncating mutations in FANCC and two with truncating mutations in BLM. Additional screening of FANCC mutation hotspot exons identified one pathogenic mutation among an additional 957 breast cancer families. Importantly, none of the deleterious mutations were identified among 464 healthy controls and are not reported in the 1,000 Genomes data. Given the rarity of Fanconi Anemia and Bloom syndrome disorders among Caucasian populations, the finding of multiple deleterious mutations in these critical DNA repair genes among high-risk breast cancer families is intriguing and suggestive of a predisposing role. Our data demonstrate the utility of intra-family exome-sequencing approaches to uncover cancer predisposition genes, but highlight the major challenge of definitively validating candidates where the incidence of sporadic disease is high, germline mutations are not fully penetrant, and individual predisposition genes may only account for a tiny proportion of breast cancer families.