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Phosphorylated neurofilament heavy subunit (pNfH) has been recently identified as a promising biomarker of disease onset and treatment efficacy in spinal muscular atrophy (SMA). This study introduces a quantitative systems pharmacology model representing the SMA pediatric scenario in the age range of 0-20 years with and without treatment with the antisense oligonucleotide nusinersen. Physiological changes typical of the pediatric age and the contribution of SMA and its treatment to the peripheral pNfH levels were included in the model by extending the equations of a previously developed mathematical model describing the neurofilament trafficking in healthy adults. All model parameters were estimated by fitting data from clinical trials that enrolled SMA patients treated with nusinersen. The data from the control group of the study was employed to build an in silico population of untreated subjects, and the parameters related to the treatment were estimated by fitting individual pNfH time series of SMA patients followed during the treatment. The final model reproduces well the pNfH levels in the presence of SMA in both the treated and untreated conditions. The results were validated by comparing model predictions with the data obtained from an additional cohort of SMA patients. The reported good predictive model performance makes it a valuable tool for investigating pNfH as a biomarker of disease progression and treatment response in SMA and for the in silico evaluation of novel treatment protocols.

The present study reports a comparison of recently described solubilizing methods, to set up a simple protocol for obtaining two-dimensional (2-D) gel electrophoresis maps of brain tissue. Different protocols were used for preparing rat brain homogenates and the resulting maps were compared by image analysis. Three different detergents, two delipidation methods, and introduction of a fractionation step based on different protein solubility in surfactants, were evaluated. When using efficient zwitterionic detergents (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, CHAPS; amidosulfobetaine 14, ASB-14), the patterns obtained by direct loading of total extracts were qualitatively overlapping with patterns obtained from fractionated samples. In contrast, a weaker nonionic agent (Nonidet P-40, NP-40) produced a different protein pattern in the collected fractions. Delipidation did not improve the results for all the different extraction methods. Immunoblots performed with antibodies recognizing cytosolic and membrane-spanning proteins, which were detected as nondegraded spots, showed that membrane proteins with intermediate molecular mass could be recovered. We suggest, as a simple and efficient method for preparing rat brain maps, the homogenization in a solution containing an efficient zwitterionic surfactant, which allows to solubilize cytosolic and membrane proteins in a single step. Alternatively, a fractionation can be carried out on samples homogenized by a weak solubilizing agent, a more labor-intensive effort resulting in a larger number of proteins on two maps.

The development of a two-dimensional (2-D) map of rat polymorphonuclear (PMN) leukocytes is here reported for the first time. The map is built up by utilizing a wide immobilized pH gradient (IPG), pH 3-10, in the first dimension and also a narrower IPG pH 4.5-8.5 gradient. In addition, the map is constructed by adopting the most recent protocols in 2-D mapping, which call for reduction and alkylation of the sample prior to the start of any electrophoretic step, including the IPG dimension. Fifty-two major protein spots have been so far identified by utilizing both matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray quadrupole (Q)-TOF mass spectrometry. A large number of house-keeping and cytoskeleton proteins were detected, together with proteins which are specific to PMN organelles or related to PMN functions such as phagocytosis and chemotaxis. The results obtained demonstrate the possibility of obtaining a single 2-D gel based proteomic map of PMN with representative proteins from different cellular compartments, also including membrane components, allowing the study of PMN protein expression on a proteome-wide scale. The aim of this project is to build an extensive database of such proteins, to be utilized for future studies where the expression of PMN proteins is used as a disease- or drug treatment marker.

The kinetics of global changes in transcription patterns during competence development in Streptococcus pneumoniae was analysed with high-density arrays. Four thousand three hundred and one clones of a S. pneumoniae library, covering almost the entire genome, were amplified by PCR and gridded at high density onto nylon membranes. Competence was induced by the addition of CSP (competence stimulating peptide) to S. pneumoniae cultures grown to the early exponential phase. RNA was extracted from samples at 5 min intervals (for a period of 30 min) after the addition of CSP. Radiolabelled cDNA was generated from isolated total RNA by random priming and the probes were hybridized to identical high-density arrays. Genes whose transcription was induced or repressed during competence were identified. Most of the genes previously known to be competence induced were detected together with several novel genes that all displayed the characteristic transient kinetics of competence-induced genes. Among the newly identified genes many have suggested functions compatible with roles in genetic transformation. Some of them may represent new members of the early or late competence regulons showing competence specific consensus sequences in their promoter regions. Northern experiments and mutational analysis were used to confirm some of the results.

3′-Substituted cephem sulfones, as well as the unsubstituted congener, were synthesized and biologically evaluated as β-lactamase inhibitors. Sodium 3′-substituted cephalosporanate sulfones 4 and 5 were prepared starting from 7-aminodeacetoxycephalosporanic acid (7-ADCA, 6) by a bromodeamination reaction, followed by reduction of the 7-halo derivative. Selective, radical bromination at C-3′ on the Δ3-isomer, followed by nucleophilic substitution and then retroisomerization at Δ2, afforded the required derivatives. 3′-Unsubstituted and 3′-acetoxycephem sulfones 2 and 3 were prepared using routine chemistry. The 3′-substituted cephem sulfone derivatives, evaluated as β-lactamase inhibitors by IC50 determinations, behave as weak inhibitors of the class D “oxacillinase” OXA1 from Escherichia coli and the class C β-lactamase of Enterobacter cloacae P99. Conversely, the 3′-unsubstituted cephem sulfone 2 was shown to be comparable to sulbactam and even more active than clavulanic acid against the latter enzyme. The activity of 2 against E. cloacae P99 was also greatly enhanced by prolonging the pre-incubation time. Considerations as to the mechanism of inhibition are also put forward.

The stereochemistry of an achiral (Diazepam) and two chiral (3-methyl and 3-succinyloxy substituted) 1,4-benzodiazepin-2-ones interacting with human serum albumin (HSA) has been investigated by making use of difference absorption (UV) and circular dichroism (CD) spectroscopies. Evidence is obtained for a higher affinity with HSA for one of the two possible conformations of the seven-membered benzodiazepine ring. The red shift revealed by the absorption difference spectrum between the free and the bound drug accounts for the CD difference spectra observed.