The value of Jamshidi core needle bone biopsy in predicting postoperative osteomyelitis in grade IV pressure ulcer patients

We present a retrospective review of 108 patients with spinal cord injury who underwent reconstruction of grade IV pressure ulcers between 1989 and 1994. Complications of reconstruction secondary to undetected osteomyelitis, namely, deep abscess and sinus tract formation, and their effect on hospital course after the flap reconstruction were quantitatively evaluated. Specifically, this study assesses whether the use of Jamshidi core needle bone biopsy allows for the accurate diagnosis, and therefore treatment, of osteomyelitis before pressure ulcer closure. Early diagnosis and treatment of osteomyelitis would presumably reduce the complication rate of reconstruction. The 108 patients in the study all underwent intraoperative Jamshidi core needle bone biopsy, and postoperative complications were evaluated by reviewers blinded to results of the biopsies. Of the 25 (23 percent) postoperative complications noted, 14 (13 percent) were attributed to underlying osteomyelitis. Patients with pressure ulcer complicated by osteomyelitis were hospitalized significantly longer than those with no osteomyelitis. On average, the former group stayed for 57 days and the latter 21 days (p < 0.001). All 14 patients who developed complications because of deep abscess and sinus tract formation had intraoperative Jamshidi core needle bone biopsy abnormality consistent with osteomyelitis (positive Jamshidi core needle bone biopsy results). The Jamshidi core needle bone biopsy compares favorably with other published modalities used to diagnose osteomyelitis, including white blood cell count, erythrocyte sedimentation rate, radiologic study, and bone biopsy culture. We propose an algorithm for the management of spinal cord injury patients with grade IV pressure ulcers. This algorithm incorporates the use of preoperative Jamshidi core needle bone biopsy to allow for the diagnosis and treatment of osteomyelitis before the flap reconstruction and to prevent complications of undiagnosed osteomyelitis after reconstruction.     

Single disulfide and linear analogues corresponding to the carboxy-terminal segment of bovine beta-defensin-2: effects of introducing the beta-hairpin nucleating sequence d-pro-gly on antibacterial activity and Biophysical properties

Mammalian defensins (alpha as well as beta forms) have a beta-hairpin structural motif spanning approximately 20 residues at the carboxy-terminal end. We have investigated the antibacterial activity and biophysical properties of synthetic peptides corresponding to the carboxy-terminal segment of bovine beta-defensin-2 (BNBD-2): VRNHVTC(1)RINRGFC(2)VPIRC(3)PGRTRQIGTC(4)FGPRIKC(5)C(6)RSW (positions of disulfide bonds are C(1)[bond]C(5), C(2)[bond]C(4), and C(3)[bond]C(6)). The parent sequence chosen was RCPGRTRQIGTIFGPRIKCRSW (P1), which spans the carboxy-terminal region of BNBD-2. Since the dipeptide sequence D-Pro-Gly favors nucleation of beta-hairpin structures even in acyclic peptides, analogues of P1 with one D-Pro-Gly at the central portion and two D-Pro-Gly segments near the N- and C-terminal ends were generated. An analogue in which GP (residues 14 and 15) in P1 was switched to PG was also synthesized. It was observed that the cyclic form as well as their linear forms exhibited antibacterial activity. Circular dichroism and theoretical studies indicated that while the beta-hairpin conformation is populated, there is conformational plasticity in the cyclic and linear peptides. The mode of bacterial killing was by membrane permeabilization. The entire mammalian defensin sequence does not appear to be essential for manifestation of antibacterial activity. Hence, short peptides corresponding to the C-terminal segments of mammalian defensins could have potential as therapeutic agents.     

Exploration of natural compounds as sources of new bifunctional scaffolds targeting cholinesterases and beta amyloid aggregation: The case of chelerythrine

The presented project started by screening a library consisting of natural and natural based compounds for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. Active compounds were chemically clustered into groups and further tested on the human cholinesterases isoforms. The aim of the presented study was to identify compounds that could be used as leads to target two key mechanisms associated with the AD’s pathogenesis simultaneously: cholinergic depletion and beta amyloid (Aβ) aggregation. Berberin, palmatine and chelerythrine, chemically clustered in the so-called isoquinoline group, showed promising cholinesterase inhibitory activity and were therefore further investigated. Moreover, the compounds demonstrated moderate to good inhibition of Aβ aggregation as well as the ability to disaggregate already preformed Aβ aggregates in an experimental set-up using HFIP as promotor of Aβ aggregates. Analysis of the kinetic mechanism of the AChE inhibition revealed chelerythrine as a mixed inhibitor. Using molecular docking studies, it was further proven that chelerythrine binds on both the catalytic site and the peripheral anionic site (PAS) of the AChE. In view of this, we went on to investigate its effect on inhibiting Aβ aggregation stimulated by AChE. Chelerythrine showed inhibition of fibril formation in the same range as propidium iodide. This approach enabled for the first time to identify a cholinesterase inhibitor of natural origin—chelerythrine—acting on AChE and BChE with a dual ability to inhibit Aβ aggregation as well as to disaggregate preformed Aβ aggregates. This compound could be an excellent starting point paving the way to develop more successful anti-AD drugs.     

Novel engineered trastuzumab conformational epitopes demonstrate in vitro and in vivo antitumor properties against HER-2/neu

Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive and has a limited duration of action, necessitating repeated administrations of the mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563-626 that comprises an extensive disulfide-bonding pattern. To delineate the binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion "promiscuous" T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformational peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563-598 and 597-626 showing greater reactivity. All epitopes were immunogenic in FVB/N mice with Abs against 597-626 and 613-626 recognizing HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting Abs which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597-626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.     

Synthesis of 9-[1-(substituted)-2-(phosphonomethoxy)ethyl]adenine derivatives as possible antiviral agents

Various C-1'-substituted acyclic N9 adenine nucleosides were prepared from 9-[(1-hydroxymethyl)(3-monomethoxytrityloxy)propyl]-N6-monomethoxytrityladenine. The hydroxymethyl was modified to the phosphonomethoxy derivative, and the 3-monomethoxytrityloxy was converted to hydroxyl, methoxy, azido, and amino. Other substituents, such as ethyl and ea-hydroxyethyl were also prepared. The resulting phosphonomethoxy derivatives were converted to prodrugs.     

Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D

The phosphoesterase (PE) domain of the bacterial DNA repair enzyme LigD possesses distinctive manganese-dependent 3′-phosphomonoesterase and 3′-phosphodiesterase activities. PE exemplifies a new family of DNA end-healing enzymes found in all phylogenetic domains. Here, we determined the structure of the PE domain of Pseudomonas aeruginosa LigD (PaePE) using solution NMR methodology. PaePE has a disordered N-terminus and a well-folded core that differs in instructive ways from the crystal structure of a PaePE•Mn²⁺• sulfate complex, especially at the active site that is found to be conformationally dynamic. Chemical shift perturbations in the presence of primer-template duplexes with 3′-deoxynucleotide, 3′-deoxynucleotide 3′-phosphate, or 3′ ribonucleotide termini reveal the surface used by PaePE to bind substrate DNA and suggest a more efficient engagement in the presence of a 3′-ribonucleotide. Spectral perturbations measured in the presence of weakly catalytic (Cd²⁺) and inhibitory (Zn²⁺) metals provide evidence for significant conformational changes at and near the active site, compared to the relatively modest changes elicited by Mn²⁺.     

Design,parallel synthesis,and crystal structures of biphenyl antithrombotics as selective inhibitors of tissue factor FVIIa complex. Part 1: Exploration of S2 pocket pharmacophores

Factor VIIa (FVIIa), a serine protease enzyme, coupled with tissue factor (TF) plays an important role in a number of thrombosis-related disorders. Inhibition of TF·FVIIa occurs early in the coagulation cascade and might provide some safety advantages over other related enzymes. We report here a novel series of substituted biphenyl derivatives that are highly potent and selective TF·FVIIa inhibitors. Parallel synthesis coupled with structure-based drug design allowed us to explore the S2 pocket of the enzyme active site. A number of compounds with IC₅₀ value of <10 nM were synthesized. The X-ray crystal structures of some of these compounds complexed with TF·FVIIa were determined and results were applied to design the next round of inhibitors. All the potent inhibitors were tested for inhibition against a panel of related enzymes and selectivity of 17,600 over thrombin, 450 over trypsin, 685 over FXa, and 76 over plasmin was achieved. Two groups, vinyl 36b and 2-furan 36ab, were identified as the optimum binding substituents on the phenyl ring in the S2 pocket. Compounds with these two substituents are the most potent compounds in this series with good selectivity over related serine proteases. These compounds will be further explored for structure–activity relationship.     

Quercetin-loaded platelets as a potential targeted therapy for glioblastoma multiforme cell line U373-MG

Over the globe, the incidence of glioblastoma multiforme (GM) is very low, that is, 1–4 cases per 100,000, but it is fatal and cancer grows very fast inside the brain tissues, namely astrocytes and oligodendrocytes. Because of the rapid growth, it is difficult to halt the dissemination of tumor in adjacent tissues. Although temozolomide (TMZ) is a currently approved standard of care, it develops resistance over the period. Therefore, there is a need to develop a novel drug delivery system. In this work, authors have developed platelets as drug delivery carriers-loaded with quercetin (QCT) for targeting GM. The effect of QCT and QCT-platelet was assessed on the U373-MG cell line. Natural human platelets were used as carriers for drug loading and drug delivery. Platelets possess an open canalicular system that allows the uptake of drug molecules in the platelet cytoplasm. The study showed that the maximum encapsulation efficiency of QCT-platelet was 93.96 ± 0.12% and the maximum drug release in 24 h was 76.26 ± 0.13% in-vitro at pH 5.5 that mimics the tumor microenvironment. In this work, there is a three-fold enhancement of solubility of QCT. The cytotoxic activity of QCT-platelets was studied in the U373-MG human astrocytoma glioblastoma cell line and the cell viability was 14.52 ± 1.53% after 48 h. Thus, platelets were proved as good carriers for therapeutic moieties and can be effectively used to target the glioblastoma tumor in the near future.     

Experimental aspects of NPY-decorated gold nanoclusters using randomized hybrid design against breast cancer cell line

Gold nanoclusters (AuNCs) are potential carrier system for bioactive like proteins and peptides used in various therapeutics against various ailments. Neuropeptide Y (NPY) is consists of 36 amino acids used to treat depression, obesity, epilepsy, and so on. but possess instability at higher temperatures causing its limited usage. The present study focused on the NPY-decorated AuNCs prepared using desolvation reduction technique and optimized through randomized hybrid design. ATR-FTIR, ¹H NMR and CD spectroscopic studies confirmed the AuNCs structure interaction with NPY. The optimized NPY-decorated AuNCs possessed 85.6 ± 2.08% of entrapment efficiency with 85.32 ± 7.55% of NPY release for 24 h. It displayed dose-dependent cell cytotoxicity, IC₅₀ value of 0.7 ± 0.05 μg mL^-1 and apoptosis of 68.48 ± 7.35% with controlled cell migration causing G0G1 cell arrest by penetrating cancer cell membrane on MCF-7 cell line. Furthermore, the AuNCs caused surface disruption of the cancerous cell further interrupting the protein synthesis by MAPK pathway leading to cell death. The AuNCs were stable for 3 months at 25 ± 2°C due to steric hindrance. Hence, NPY-decorated AuNCs were found to be effective on MCF-7 cell line with a significant anti-apoptotic effect, further emerging as a novel therapeutic delivery system in the management of breast cancer.     

Pulmonary arterial hypertension: a disease of tethers, SNAREs and SNAPs?

Histological and electron microscopic studies over the past four decades have highlighted "plump," "enlarged" endothelial, smooth muscle, and fibroblastic cellular elements with increased endoplasmic reticulum, Golgi stacks, and vacuolation in pulmonary arterial lesions in human and in experimental (hypoxia and monocrotaline) pulmonary arterial hypertension. However, the contribution of disrupted intracellular membrane trafficking in the pathobiology of this disease has received insufficient attention. Recent studies suggest a pathogenetic role of the disruption of intracellular trafficking of vasorelevant proteins and cell-surface receptors in the development of this disease. The purpose of this essay is to highlight the molecular regulation of vesicular trafficking by membrane tethers, SNAREs and SNAPs, and to suggest how their dysfunction, directly and/or indirectly, might contribute to development of pulmonary arterial hypertension in experimental models and in humans, including that due to mutations in bone morphogenetic receptor type 2.