Bacterial secretion of the Fab fragment of a mouse monoclonal IgM that reacts with IgG variable regions

In this report, we describe the expression system that enabledus to produce in Escherichia coli the Fab fragment of a mouseIgM that has previously been shown to inhibit the binding ofIgG to autoantigens by interacting with their variable regions.In our system, both light chain and heavy chain fragments wereput under the control of the malE promoter. The light chainwas fused to the MalE signal sequence, while the heavy chainvariable and first constant region were fused to the alkalinephosphatase signal sequence. In this system, after inductionof the promoter with maltose, the Fab fragment could be detectedin a periplasmic extract of the bacteria by Western blottingand also by ELISA. This Fab fragment was purified on a goatanti-mouse immunoglobulin immunoadsorbent and biotinylated.The Fab fragment produced by E.coli reacted with the trinitrophenyl(TNP) hapten and F(ab')₂ fragments of mouse IgG and these reactivitiescould be specifically inhibited by the corresponding solubleantigens. The dissociation constants of this Fab were 1.65 x10^–6 M for TNP and 5 x 10^–6 M for IgG F(ab')₂ fragments,indicating that the affinity of the Fab fragment compared withthat of the whole IgM molecule was similar for TNP but was lowerfor IgG F(ab')₂ fragments     

Potent,Metabolically Stable 2‐Alkyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐adenines as Adenosine A2A Receptor Ligands

Inhibition of adenosine A_2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson’s disease (PD). We previously identified the triazolo‐9H‐purine, ST1535, as a potent A_2AR antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω‐1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω‐1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A_2AR was determined. Two compounds, (2‐(3,3‐dimethylbutyl)‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐6‐amine ( 3 b ) and 4‐(6‐amino‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐2‐yl)‐2‐methylbutan‐2‐ol ( 3 c ), exhibited good affinity against A_2AR (K_i=0.4 nM and 2 nM , respectively) and high in vitro metabolic stability (89.5 % and 95.3 % recovery, respectively, after incubation with HLM for two hours).     

Characterisation of CYP102A25 from Bacillus marmarensis and CYP102A26 from Pontibacillus halophilus: P450 Homologues of BM3 with Preference towards Hydroxylation of Medium‐Chain Fatty Acids

Cytochrome P450 monooxygenases are highly desired biocatalysts owing to their ability to catalyse a wide variety of chemically challenging C?H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self‐sufficient proteins consisting of a haem and FMN‐FAD reductase domain fused in a single‐component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω?1, ω?2 and ω?3 hydroxy acids. These monooxygenases have potential applications in biotechnology; however, their substrate range is still limited and there is a continued need to add diversity to this class of biocatalysts. Herein, we present the characterisation of two new members of this class of enzymes, CYP102A25 (BMar) from Bacillus marmarensis and CYP102A26 (PHal) from Pontibacillus halophilus, both of which express readily in a recombinant bacterial host. BMar exhibits the highest activity toward myristic acid and shows moderate activity towards unsaturated fatty acids. PHal exhibits broader activity towards mid‐chain‐saturated (C₁₄–C₁₈) and unsaturated fatty acids. Furthermore, PHal shows good regioselectivity for the hydroxylation of myristic acid, targeting the ω?2 position for C?H activation.     

Comparison between ozonation and the OSA process: analysis of excess sludge reduction and biomass activity in two different pilot plants

The excess biomass produced during biological treatment of municipal wastewater represents a major issue worldwide, as its disposal implies environmental, economic and social impacts. Therefore, there has been a growing interest in developing technologies to reduce sludge production. The main proposed strategies can be categorized according to the place inside the wastewater treatment plant (WWTP) where the reduction takes place. In particular, sludge minimization can be achieved in the wastewater line as well as in the sludge line. This paper presents the results of two pilot scale systems, to evaluate their feasibility for sludge reduction and to understand their effect on biomass activity: (1) a pilot plant with an ozone contactor in the return activated sludge (RAS) stream for the exposition of sludge to a low ozone dosage; and (2) an oxic-settling-anaerobic (OSA) process with high retention time in the anaerobic sludge holding tank have been studied. The results showed that both technologies enabled significant excess sludge reduction but produced a slight decrease of biomass respiratory activity.     

Biological Effects of Cyclin-Dependent Kinase Inhibitors Ribociclib,Palbociclib and Abemaciclib on Breast Cancer Bone Microenvironment

The CDK4/6 inhibitors (CDKi) palbociclib, ribociclib, and abemaciclib are currently approved in combination with anti-estrogen therapy for the treatment of advanced and/or metastatic hormone receptor-positive/HER2-neu-negative breast cancer patients. Given the high incidence of bone metastases in this population, we investigated and compared the potential effects of palbociclib, ribociclib, and abemaciclib on the breast cancer bone microenvironment. Primary osteoclasts (OCs) and osteoblasts (OBs) were obtained from human monocyte and mesenchymal stem cells, respectively. OC function was evaluated by tartrate-resistant acid phosphatase assay and real-time PCR; OB activity was assessed by an alizarin red assay. OB/breast cancer co-culture models were generated via the seeding of MCF-7 cells on a layer of OBs, and tumor cell proliferation was analyzed using flow cytometry. Here, we showed that ribociclib, palbociclib, and abemaciclib exerted similar inhibitory effects on the OC differentiation and expression of bone resorption markers without affecting OC viability. On the other hand, the three CDKi did not affect the ability of OB to produce bone matrix, even if the higher doses of palbociclib and abemaciclib reduced the OB viability. In OB/MCF-7 co-culture models, palbociclib demonstrated a lower anti-tumor effect than ribociclib and abemaciclib. Overall, our results revealed the direct effects of CDKi on the tumor bone microenvironment, highlighting differences potentially relevant for clinical practice.     

The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.     

Cranberry and Recurrent Cystitis: More than Marketing?

Epidemiologic studies indicate that millions of people suffer from recurrent cystitis, a pathology requiring antibiotic prophylaxis and entailing high social costs. Cranberry is a traditional folk remedy for cystitis and, which, in the form of a variety of products and formulations has over several decades undergone extensive evaluation for the management of urinary tract infections (UTI). The aim of this retrospective study is to summarize and review the most relevant and recent preclinical and clinical studies on cranberries for the treatment of UTIs. The scientific literature selected for this review was identified by searches of Medline via PubMed. A variety of recent experimental evidence has shed light on the mechanism underlying the anti-adhesive properties of proanthrocyanidins, their structure–activity relationships, and pharmacokinetics. Analysis of clinical studies and evaluation of the cranberry efficacy/safety ratio in the prevention of UTIs strongly support the use of cranberry in the prophylaxis of recurrent UTIs in young and middle-aged women. However, evidence of its clinical use among other patients remains controversial.     

Passive adaptation or active engagement? The challenges of Housing First internationally and in the Italian case

In recent years, a peculiar homelessness policy that goes under the name of ‘Housing First’ has become increasingly popular all over the world. Epitomising a quintessential case of policy-mobility, Housing First can today be considered an heterogeneous assemblage of experiences and approaches that sometimes have little in common with each other. Introducing and commenting upon this heterogeneity, the paper critically analyses why and how Housing First has become a planetary success and what are the issues at stake with its widespread implementation. If recent scholarship published in this journal has granted us a fine understanding of Housing First’s functioning in the US, this paper offers something currently absent from the debate: a nuanced and critical understanding of the ambiguities related to the international success of this policy, with specific references to the challenges associated to its translation in the Italian case.     

SmartK: Smart cards in operating systems at kernel level

A smart card is a tamper-resistant miniature computer that performs some basic computations on input a secret information. So far, smart cards have been widely used for securing many digital transactions (e.g., pay television, ATM machines).We focus on the implementation of operating system security services leveraging on smart cards. This very challenging feature allows one to personalize some functionalities of the operating system by simply changing a smart card. Current solutions for integrating smart card features in operating system services require at least a partial execution of some of the operating system functionalities at “user level”. Unfortunately, system functionalities built on top of components lying at both kernel and user levels may negatively affect the overall system security, due to the introduction of multiple points of failure.In this work, we present the design and implementation of SmartK: a framework that integrates features of smart cards uniquely in the Linux kernel. In order to validate our approach, we propose a host of enhancements to the Linux operating system built on top of SmartK: 1) in-kernel clients' authentication with Kerberos; 2) execution of trusted code; 3) key management in secure network filesystems.In particular, we present an experimental Linux OS distribution (SalSA), which addresses the security issues related to downloading packages and to updating an operating system through the Internet.