Enantiomerically pure tetrahydroquinoline derivatives as in vivo potent antagonists of the glycine binding site associated to the NMDA receptor

To identify neuroprotective agents after stroke, new substituted tetrahydroquinoline derivatives were designed as antagonists of the glycine binding site associated to the NMDA receptor, satisfying the key pharmacophoric requirements. In particular, the racemate 3c exhibited outstanding in vivo activity in the MCAo model in rats, when given iv both pre- and post-ischemia. Pure enantiomers 3c-(+) and 3c-(-) have been prepared following an original synthetic route. Despite the significant difference of activity observed in vitro, they shown similar neuroprotective profile in the MCAo model in rats.     

Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase.

The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site-directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pastoris cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lipl of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.     

Reliable and Accurate CD4+ T Cell Count and Percent by the Portable Flow Cytometer CyFlow MiniPOC and “CD4 Easy Count Kit-Dry”, as Revealed by the Comparison with the Gold Standard Dual Platform Technology

Background

An accurate and affordable CD4+ T cells count is an essential tool in the fight against HIV/AIDS. Flow cytometry (FCM) is the “gold standard” for counting such cells, but this technique is expensive and requires sophisticated equipment, temperature-sensitive monoclonal antibodies (mAbs) and trained personnel. The lack of access to technical support and quality assurance programs thus limits the use of FCM in resource-constrained countries. We have tested the accuracy, the precision and the carry-over contamination of Partec CyFlow MiniPOC, a portable and economically affordable flow cytometer designed for CD4+ count and percentage, used along with the “CD4% Count Kit-Dry”.

Materials and Methods

Venous blood from 59 adult HIV+ patients (age: 25–58 years; 43 males and 16 females) was collected and stained with the “MiniPOC CD4% Count Kit-Dry”. CD4+ count and percentage were then determined in triplicate by the CyFlow MiniPOC. In parallel, CD4 count was performed using mAbs and a CyFlow Counter, or by a dual platform system (from Beckman Coulter) based upon Cytomic FC500 (“Cytostat tetrachrome kit” for mAbs) and Coulter HmX Hematology Analyzer (for absolute cell count).

Results

The accuracy of CyFlow MiniPOC against Cytomic FC500 showed a correlation coefficient (CC) of 0.98 and 0.97 for CD4+ count and percentage, respectively. The accuracy of CyFlow MiniPOC against CyFlow Counter showed a CC of 0.99 and 0.99 for CD4 T cell count and percentage, respectively. CyFlow MiniPOC showed an excellent repeatability: CD4+ cell count and percentage were analyzed on two instruments, with an intra-assay precision below ±5% deviation. Finally, there was no carry-over contamination for samples at all CD4 values, regardless of their position in the sequence of analysis.

Conclusion

The cost-effective CyFlow MiniPOC produces rapid, reliable and accurate results that are fully comparable with those from highly expensive dual platform systems.     

Automatic detection of surface EMG activation timing using a wavelet transform based method

The problem of the identification of the muscle contraction timing by using surface electromyographic signal is addressed. The timing detection of the muscular activation in dynamic conditions has a real clinical diagnostic impact. Widely used single threshold methods still rely on the experience of the operator in manually setting that threshold. A new approach to detect the muscular activation intervals, that is based on discontinuities detection in the wavelet domain, is proposed. Accuracy and precision of the algorithm were assessed by using a set of simulated signals obtaining values lower than 11.0 and 8.7 ms for biases and standard deviations of the estimation, respectively. Moreover an experimental application of the algorithm was carried out recruiting a population of 10 able-bodied subjects and processing the myoelectric signals recorded from the lower limb during an isokinetic exercise. The algorithm was able to reveal correctly the timing of muscular activation with performance comparable to the state-of-the-art methods. The detection algorithm is automatic and user-independent, it manages the detection of both onset and offset activation, it can be fruitfully applied even in presence of noise and, therefore, it can be used also by unskilled operators.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

Three-dimensional in vitro follicle growth: overview of culture models,biomaterials, design parameters and future directions

In vitro ovarian follicle culture is a new frontier in assisted reproductive technology with tremendous potential, especially for fertility preservation. Folliculogenesis within the ovary is a complex process requiring interaction between somatic cell components and the oocyte. Conventional two-dimensional culture on tissue culture substrata impedes spherical growth and preservation of the spatial arrangements between oocyte and surrounding granulosa cells. Granulosa cell attachment and migration can leave the oocyte naked and unable to complete the maturation process. Recognition of the importance of spatial arrangements between cells has spurred research in to three-dimensional culture system. Such systems may be vital when dealing with human primordial follicles that may require as long as three months in culture. In the present work we review pertinent aspects of in vitro follicle maturation, with an emphasis on tissue-engineering solutions for maintaining the follicular unit during the culture interval. We focus primarily on presenting the various 3-dimensional culture systems that have been applied for in vitro maturation of follicle:oocyte complexes. We also try to present an overview of outcomes with various biomaterials and animal models and also the limitations of the existing systems.