Intravital three-photon microscopy allows visualization over the entire depth of mouse lymph nodes.

Intravital confocal microscopy and two-photon microscopy are powerful tools to explore the dynamic behavior of immune cells in mouse lymph nodes (LNs), with penetration depth of ~100 and ~300 µm, respectively. Here, we used intravital three-photon microscopy to visualize the popliteal LN through its entire depth (600-900 µm). We determined the laser average power and pulse energy that caused measurable perturbation in lymphocyte migration. Long-wavelength three-photon imaging within permissible parameters was able to image the entire LN vasculature in vivo and measure CD8+ T cells and CD4+ T cell motility in the T cell zone over the entire depth of the LN. We observed that the motility of naive CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth. As such, intravital three-photon microscopy had the potential to examine immune cell behavior in the deeper regions of the LN in vivo.

Single versus double flap approach in periodontal regenerative treatment.

AIM: to compare the outcomes of a regenerative strategy based on recombinant human platelet-derived growth factor-BB (rhPDGF-BB, 0.3 mg/ml) and ß-tricalcium phosphate (ß-TCP) in the treatment of intraosseous defects accessed with the Single Flap Approach (SFA) versus Double Flap Approach based on papilla preservation techniques (DFA). MATERIALS AND METHODS: Fifteen and 13 defects, randomly assigned to SFA or DFA, respectively, were grafted with rhPDGF-BB + ß-TCP. Probing parameters were assessed before and 6 months after surgery. Pain (VAS(pain)) was self-reported using a visual analogue scale. RESULTS: Twelve SFA sites and DFA 6 sites showed complete flap closure at 2 weeks post-surgery. No significant differences in 6-month changes in probing parameters and radiographic defect fill were found between groups. Significantly lower VAS(pain) was observed in SFA group compared to DFA group at day +1, +2 and +6. A significantly greater number of analgesics were consumed in the DFA group compared to the SFA group at day +1. CONCLUSIONS: When combined with rhPDGF-BB and ß-TCP, the SFA may result in similar clinical outcomes, better quality of early wound healing, and lower pain and consumption of analgesics during the first postoperative days compared to the DFA.

Contribution of hydrogen bonds to protein stability.

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.

Solution structure and calcium-binding properties of EF-hands 3 and 4 of calsenilin.

Calsenilin is a member of the recoverin branch of the EF-hand superfamily that is reported to interact with presenilins, regulate prodynorphin gene expression, modulate voltage-gated Kv4 potassium channel function, and bind to neurotoxins. Calsenilin is a Ca+2-binding protein and plays an important role in calcium signaling. Despite its importance in numerous neurological functions, the structure of this protein has not been reported. In the absence of Ca+2, the protein has limited spectral resolution that increases upon the addition of Ca+2. Here, we describe the three-dimensional solution structure of EF-hands 3 and 4 of calsenilin in the Ca+2-bound form. The Ca+2-bound structure consists of five alpha-helices and one two-stranded antiparallel beta-sheet. The long loop that connects EF hands 3 and 4 is highly disordered in solution. In addition to its structural effects, Ca+2 binding also increases the protein's propensity to dimerize. These changes in structure and oligomerization state induced upon Ca+2 binding may play important roles in molecular recognition during calcium signaling.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

Mannose-6-phosphate/insulin-like growth factor II receptor expression and tumor development.

The mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR) is a multi-functional transmembrane glycoprotein whose major function is to bind and transport M6P-bearing glycoproteins from the trans-Golgi network or the cell surface to lysosomes. The cell surface M6P/IGF-IIR also bind and internalizes the insulin-like growth factor II. The receptor gene is considered a "candidate" tumor suppressor gene. The phenotypic consequences of loss of M6P/IGF-IIR through somatic mutation are potentially very complex since M6P/IGF-IIR has a number of roles in cellular physiology. Loss of function mutations in M6P/IGF-IIR gene could contribute to multi-step carcinogenesis. In the light of the multi-functional cellular potential roles of the M6P/IGF-IIR the purpose of this review is to highlight some recent data concerning its normal functions and the potential role of its loss in tumor pathophysiology with the aim to try to clarify the possible underlying mechanisms of its involvement in tumor development.

Transforming the work of early-stage drug discovery through bioprocess informatics.

Drug discovery has historically advanced by synergy and chance. These are proving insufficient to meet the needs of the marketplace and the demands of modern medicine. We describe our strategic approaches to building and employing flexible informatics tools to transform and improve the workflows and efficiencies of the early-stages of target development in drug discovery. We contrast our approach to strategies that have recently evolved at startup biotechnology companies who use similar technological approaches to drug development but who are less encumbered by precedent and history.

Improvement of exogenous DNA nuclear importation by nuclear localization signal-bearing vectors: a promising way for non-viral gene therapy?

Several vectors have been developed in order to target genes to specific cells. Virus-based vectors lead to a high transfection efficiency in vitro, but display important disadvantages such as pathological risks, which they expose to patients. Plasmid-associated chemical vectors lack these disadvantages, but allow only a very low efficiency of transgene expression. Most of the non-viral-based gene transfer techniques developed until now mainly focused their efforts to overcome the problem of DNA entry into the cell. Some recent works, however, have begun to investigate the nucleus entry problem and suggest that the trafficking of DNA from cytosol to the nucleus may be improved by using the nuclear localization signal (NLS) found in some nuclear proteins. If the vector contains one or several NLS, either as covalently or non-covalently DNA-linked peptides, a competition may take place between the rate of dissociation of the DNA-vector complexes and the rate of loading of the complexes to the NLS-mediated nucleus importation machinery. This equilibrium may be displaced towards the importation pathway by the use of NLS-bearing proteins instead of peptides. The possibility of recruiting normal endogenous cellular pathways of nuclear uptake to promote entry of exogenously applied DNA through the nuclear pore complex would, thus, seem promising. Nevertheless, attempts to improve the transport of DNA to the nucleus through the use of NLSs have achieved limited success. Although these systems show improved transgene expression, little is known about how they function in transfected cells, and the optimal formulation for gene expression is yet to be determined.

Lid-wiper epitheliopathy and dry-eye symptoms in contact lens wearers.

PURPOSE: To evaluate whether dry-eye symptoms are associated with epitheliopathy of that portion of the upper eyelid marginal conjunctiva-the lid wiper-that wipes the ocular, or contact lens surface, during blinking. METHODS: Subjects were divided into two groups based on the presence or absence of dry-eye symptoms. The lid wiper of asymptomatic (n=75) and symptomatic (n=30) soft contact lens wearers was examined, following the instillation of fluorescein and rose bengal dyes. Lid-wiper staining was graded zero to 3. RESULTS: Eighty percent of the symptomatic subjects displayed lid-wiper staining compared to 13% of the asymptomatic subjects. The difference in staining between the two groups was significant (P<0.0001). Of the symptomatic subjects, 20% showed no staining; 26.6%, grade 1 staining; 36.6%, grade 2; and 16.6% showed grade 3 staining. Of the asymptomatic subjects, 87% exhibited no staining; 9%, grade 1 staining; 3%, grade 2; and 1% showed grade 3 staining. CONCLUSIONS: This study describes a new clinical condition, lid-wiper epitheliopathy, an alteration of the epithelium of that portion of the marginal conjunctiva of the upper eyelid that wipes the ocular surface, diagnosed by staining with fluorescein and rose bengal dyes.