Structure and Inhibitor Specificity of L,D-Transpeptidase (LdtMt2) from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and Antibiotic Resistance: Calcium Binding Promotes Dimer Formation

The final step of peptidoglycan (PG) synthesis in all bacteria is the formation of cross-linkage between PG-stems. The cross-linking between amino acids in different PG chains gives the peptidoglycan cell wall a 3-dimensional structure and adds strength and rigidity to it. There are two distinct types of cross-linkages in bacterial cell walls. D,D-transpeptidase (D,D-TPs) generate the classical 4?3 cross-linkages and the L,D-transpeptidase (L,D-TPs) generate the 3?3 non-classical peptide cross-linkages. The present study is aimed at understanding the nature of drug resistance associated with L,D-TP and gaining insights for designing novel antibiotics against multi-drug resistant bacteria. Penicillin and cephalosporin classes of β-lactams cannot inhibit L,D-TP function; however, carbapenems inactivate its function. We analyzed the structure of L,D-TP of Mycobacterium tuberculosis in the apo form and in complex with meropenem and imipenem. The periplasmic region of L,D-TP folds into three domains. The catalytic residues are situated in the C-terminal domain. The acylation reaction occurs between carbapenem antibiotics and the catalytic Cys-354 forming a covalent complex. This adduct formation mimics the acylation of L,D-TP with the donor PG-stem. A novel aspect of this study is that in the crystal structures of the apo and the carbapenem complexes, the N-terminal domain has a muropeptide unit non-covalently bound to it. Another interesting observation is that the calcium complex crystallized as a dimer through head and tail interactions between the monomers.     

Informative value of clinical research on multislice computed tomography in the diagnosis of coronary artery disease: A systematic review

Multislice Spiral Computed Tomography (MSCT) is an emerging non-invasive diagnostic modality to detect coronary artery disease, which may alter diagnostic pathways and change the current clinical role of conventional coronary angiography.

Aims

To retrieve and critically assess information from the available literature on MSCT (≥ 16-slice) concerning its diagnostic accuracy, safety, applicability, clinical impact and cost-effectiveness.

Methods and results

Articles published between January 2002 and March 2007 were identified through searches of the Cochrane Library, MEDLINE, and other websites of manufacturers, cardiac professional societies, guidelines and abstracts from conference meetings.We identified 1768 potentially relevant articles: 262 out of these were considered eligible for full evaluation and 150 were selected (57 assessed diagnostic accuracy, 130 applicability, 103 safety, 1 clinical impact and none cost-effectiveness).The pre test probability of coronary artery disease was 56.7% (95% Confidence Interval: 55.1%–58.3%). A positive MSCT finding (pooled LR+: 5.4 (4.4–6.7)) increased the probability of CAD to 87.7% (84.3%–90.3%), whereas a negative MSCT result (pooled LR−: 0.09 (0.07–0.12)) reduced the probability of CAD to 10.7% (7.9%–14.4%).

Conclusions

MSCT is a promising technology for the assessment of coronary artery stenosis. However, the available literature is of limited value in providing guidance to support the development of policies for its appropriate utilization in clinical practice.     

Synthesis and evaluation of technetium-99m- and rhenium-labeled inhibitors of the prostate-specific membrane antigen (PSMA)

The prostate-specific membrane antigen (PSMA) is increasingly recognized as a viable target for imaging and therapy of cancer. We prepared seven (99m)Tc/Re-labeled compounds by attaching known Tc/Re chelating agents to an amino-functionalized PSMA inhibitor (lys-NHCONH-glu) with or without a variable length linker moiety. K i values ranged from 0.17 to 199 nM. Ex vivo biodistribution and in vivo imaging demonstrated the degree of specific binding to engineered PSMA+ PC3 PIP tumors. PC3-PIP cells are derived from PC3 that have been transduced with the gene for PSMA. Despite demonstrating nearly the lowest PSMA inhibitory potency of this series, [(99m)Tc(CO)3( L1)] (+) ( L1 = (2-pyridylmethyl)2N(CH2) 4CH(CO2H)NHCO-(CH2) 6CO-NH-lys-NHCONH-glu) showed the highest, most selective PIP tumor uptake, at 7.9 +/- 4.0% injected dose per gram of tissue at 30 min postinjection. Radioactivity cleared from nontarget tissues to produce a PIP to flu (PSMA-PC3) ratio of 44:1 at 120 min postinjection. PSMA can accommodate the steric requirements of (99m)Tc/Re complexes within PSMA inhibitors, the best results achieved with a linker moiety between the epsilon amine of the urea lysine and the chelator.     

Combining high-frequency oscillatory ventilation and recruitment maneuvers in adults with early acute respiratory distress syndrome: the Treatment with Oscillation and an Open Lung Strategy (TOOLS) Trial pilot study

OBJECTIVE: To determine the safety, feasibility, and lung-recruitment efficacy of an explicit ventilation protocol combining high-frequency oscillatory ventilation and recruitment maneuvers. DESIGN: Prospective, multiple-center, single-intervention pilot study. SETTING: Four university-affiliated intensive care units. PATIENTS: Twenty-five patients with early acute respiratory distress syndrome and severe oxygenation failure. INTERVENTIONS: Patients were transitioned from standardized conventional ventilation to high-frequency oscillatory ventilation beginning with an initial cycle of up to three sustained inflation recruitment maneuvers (40 cm H2O x 40 secs), followed by a decremental titration of Fio2 and then mean airway pressure. Recruitment maneuvers were repeated for hypoxemia and routinely at least twice daily if the Fio2 was >0.4. A specific protocol was used for weaning high-frequency oscillatory ventilation, for transitioning to conventional ventilation, and for judging intolerance of conventional ventilation whereby patients should be put back on high-frequency oscillatory ventilation. MEASUREMENTS AND MAIN RESULTS: Patients (median [interquartile range] Acute Physiology and Chronic Health Evaluation II, 24 [19-32]; age, 50 [41-64]) were enrolled after 13 (range, 6-51) hrs of conventional ventilation. Following the initial cycle of recruitment, the mean (+/-sd) Pao2/Fio2 increased significantly compared with standardized conventional ventilation (200 +/- 117 vs. 92 +/- 36 mm Hg, p < .001). After a mean of 12 hrs of high-frequency oscillatory ventilation, the mean Fio2 was significantly reduced compared with prestudy levels (0.5 +/- 0.2 vs. 0.9 +/- 0.1, p < .001). A median of seven (four to 11) recruitment maneuvers was performed per patient over the study period, with only eight of 244 (3.3%) being aborted. Six of 19 patients transitioned to conventional ventilation (32%) were deemed intolerant and were switched back to high-frequency oscillatory ventilation. Protocol adherence was excellent with documented rates >90%. CONCLUSIONS: The combination of high-frequency oscillatory ventilation and recruitment maneuvers resulted in rapid and sustained improvement in oxygenation, likely through lung recruitment. This explicit high-frequency oscillatory ventilation protocol appears well tolerated, feasible, and physiologically sound.     

Utility of routine chest radiographs in a medical–surgical intensive care unit: a quality assurance survey

Objective  

To determine the utility of routine chest radiographs (CXRs) in clinical decision-making in the intensive care unit (ICU).     

BK nephropathy in pediatric hematopoietic stem cell transplant recipients

Abstract:  BK nephropathy is a known cause of renal insufficiency in kidney transplant recipients. Activation of the polyoma virus may also occur in the native kidneys of non-renal allograft recipients. BK nephropathy has only been reported in a few patients after HCT, most being adult patients, and the single reported pediatric case had evidence of hemorrhagic cystitis. The response to antiviral therapy also seems to differ widely. Here, we describe two cases of BK nephropathy in the native kidneys of HCT recipients exposed to high levels of immunosuppression because of GVHD. Neither of our patients had any evidence of hemorrhagic cystitis. We present definitive renal pathology and detailed chronological evidence of the rising serum creatinine with simultaneous serum and urine BK PCR titers. In one of our cases, antiviral therapy did not seem beneficial as documented by continued renal dysfunction and elevated serum/urine BK PCR titers. Based on our report, intense immunosuppression in pediatric HCT recipients seems to be involved in the activation of BK virus and BK nephropathy should be suspected even in the absence of hematuria in HCT recipients with unexplained renal dysfunction.     

Coronary calcium score as gatekeeper for 64-slice computed tomography coronary angiography in patients with chest pain: per-segment and per-patient analysis

We sought to investigate the performance of 64-slice CT in symptomatic patients with different coronary calcium scores. Two hundred patients undergoing 64-slice CT coronary angiography for suspected coronary artery disease were enrolled into five groups based on Agatston calcium score using the Mayo Clinic risk stratification: group 1: score 0, group 2: score 1–10, group 3: score 11–100, group 4: score 101–400, and group 5: score > 401. Diagnostic accuracy for the detection of significant (≥50% lumen reduction) coronary artery stenosis was assessed on a per-segment and per-patient base using quantitative coronary angiography as the gold standard. For groups 1 through 5, sensitivity was 97, 96, 91, 90, 92%, and specificity was 99, 98, 96, 88, 90%, respectively, on a per-segment basis. On a per-patient basis, the best diagnostic performance was obtained in group 1 (sensitivity 100% and specificity 100%) and group 5 (sensitivity 95% and specificity 100%). Progressively higher coronary calcium levels affect diagnostic accuracy of CT coronary angiography, decreasing sensitivity and specificity on a per-segment base. On a per-patient base, the best results in terms of diagnostic accuracy were obtained in the populations with very low and very high cardiovascular risk. Authors have no financial conflict of interest. Neither this paper nor any of its content has not been submitted to other journals.     

Panaxadiol, a purified ginseng component, enhances the anti-cancer effects of 5-fluorouracil in human colorectal cancer cells

Purpose  Colorectal cancer is a major cause of morbidity and mortality for cancer worldwide. Although 5-fluorouracil (5-FU) is one of the most widely used chemotherapeutic agents in first-line therapy for colorectal cancer, serious side effects limit its clinical usefulness. Panaxadiol (PD) is the purified sapogenin of ginseng saponins, which exhibit anti-tumor activity. In this study, we investigated the possible synergistic anti-cancer effects of PD and 5-FU on a human colorectal cancer cell line, HCT-116. Methods  Cell viability was evaluated by an MTS cell proliferation assay. Morphological observation was performed by crystal violet cell viability staining assay. Cell cycle distribution and apoptotic effects were analyzed by flow cytometry after staining with PI/RNase or Annexin V/PI. Results  Cell growth was markedly suppressed in HCT-116 cells treated by 5-FU (20–100 μM) for 24 or 48 h with time-dependent effects. The significant suppression on HCT-116 cell proliferation was observed after treatment with PD (25 μM) for 24 and 48 h. Panaxadiol (25 μM) markedly (P < 0.05) enhanced the anti-proliferative effects of 5-FU (5, 10, 20 μM) on HCT-116 cells compared to single treatment of 5-FU for 24 and 48 h. Flow cytometric analysis on DNA indicated that PD and 5-FU selectively arrested cell cycle progression in the G1 phase and S phase (P < 0.01), respectively, compared to the control condition. Combination use of 5-FU with PD significantly (P < 0.001) increased cell cycle arrest in the S phase compared to that treated by 5-FU alone. The combination of 5-FU and PD significantly enhanced the percentage of apoptotic cells when compared with the corresponding cell groups treated by 5-FU alone (P < 0.001). Conclusions  Panaxadiol enhanced the anti-cancer effects of 5-FU on human colorectal cancer cells through the regulation of cell cycle transition and the induction of apoptotic cells.     

Characterization of a targeted nanoparticle functionalized with a urea-based inhibitor of prostate-specific membrane antigen (PSMA)

Polymeric nanoparticles represent a form of targeted therapy due to their ability to passively accumulate within the tumor interstitium via the enhanced permeability and retention (EPR) effect. We used a combined approach to decorate the surface of a nanoparticle with a urea-based small-molecule peptidomimetic inhibitor of prostate specific membrane antigen (PSMA). PSMA is expressed by normal and malignant prostate epithelial cells and by the neovasculature of almost all solid tumors. This strategy takes advantage of both the avidity of the functionalized nanoparticle for binding to PSMA and the ability of the nanoparticle to be retained for longer periods of time in the tumor due to enhanced leakage via EPR into the tumor interstitium. As an initial step to introducing the targeting moiety, the amino terminus of the small-molecule PSMA inhibitor was conjugated to PEG (M(n) 3400) bearing an activated carboxyl group to obtain a PEGylated inhibitor. Studies undertaken using a radiolabeled PSMA-substrate based assay established that the PEGylated inhibitor had an IC(50) value similar to the uncomplexed inhibitor. Subsequently, nanoparticles loaded with docetaxel were formulated using a mixture of poly(lactide-beta-ethylene glycol-beta-lactide) and PSMA-inhibitor bound alpha-amino-omega-hydroxy terminated poly (ethylene glycol-beta-epsilon-caprolactone). In vitro studies using these nanoparticles demonstrated selective cytotoxicity against PSMA-producing cells. Binding of fluorescently labeled PSMA-targeted particles to PSMA-producing cells has also been directly observed using fluorescence microscopy and observed in secondary fashion using a PSMA substrate based enzyme inhibition assay. Ongoing in vivo studies address the localization, activity and toxicity of these targeted nanoparticles against PSMA-producing human prostate tumor xenografts.