Screen for inhibitors of the coupled transglycosylase-transpeptidase of peptidoglycan biosynthesis in Escherichia coli

Class A high-molecular-weight penicillin-binding protein 1a (PBP1a) and PBP1b of Escherichia coli have both transglycosylase (TG) and transpeptidase (TP) activity. These enzymes are difficult to assay, since their substrates are difficult to prepare. We show the activity of PBP1a or PBP1b can be measured in membranes by cloning the PBP into an E. coli ponB::Spcr strain. Using this assay, we show that PBP1a is approximately 10-fold more sensitive to penicillin than PBP1b and that the 50% inhibitory concentration (IC50) of moenomycin, a TG inhibitor, is approximately 10-fold higher in the PBP transformants than in wild-type membranes; this increase in IC50 in transformants can be used to test the specificity of test compounds for inhibition of the TG. Alternatively, the coupled TG-TP activity of PBP1b can be directly measured in a two-step microplate assay. In the first step, radiolabeled lipid II, the TG substrate, was made in membranes of the E. coli ponB::Spcr strain by incubation with the peptidoglycan sugar precursors. In the second step, the TG-TP activity was assayed by adding a source of PBP1b to the membranes. The coupled TG-TP activity converts lipid II to cross-linked peptidoglycan, which was specifically captured by wheat germ agglutinin-coated scintillation proximity beads in the presence of 0.2% Sarkosyl (B. Chandrakala et al., Antimicrob. Agents Chemother. 48:30-40, 2004). The TG-TP assay was inhibited by penicillin and moenomycin as expected. Surprisingly, tunicamycin and nisin also inhibited the assay, and paper chromatography analysis revealed that both inhibited the transglycosylase. The assay can be used to screen for novel antibacterial agents.     

Presence of copper- and zinc-containing superoxide dismutase in commensal Haemophilus haemolyticus isolates can be used as a marker to discriminate them from nontypeable H. influenzae isolates

Respiratory isolates of Haemophilus haemolyticus are regularly misclassified as nontypeable (NT) Haemophilus influenzae due to an aberrant hemolytic reaction on blood agar, with implications for treatment. The presence of sodC or its cognate protein, copper-zinc superoxide dismutase, can distinguish respiratory isolates of H. haemolyticus from NT H. influenzae with 100% accuracy.     

Endoscopic craniofacial resection for pediatric esthesioneuroblastoma

Pediatric esthesioneuroblastomas are rare tumors with only about 100 reported cases. Standard surgical treatment is the open craniofacial resection. Pre-treatment imaging is critical for planning, and surgery remains the primary treatment modality followed by adjuvant therapy. We report a 12-year-old female who underwent endoscopic-assisted resection of an extensively calcified Kadish C and Dulguerov–Calcaterra T3N0M0 esthesioneuroblastoma. Following radiation therapy, she is disease-free 18 months post-treatment. Although there are few reported cases in pediatric patients, our case supports the use of endoscopic endonasal approaches for select pediatric skull base malignancies.     

Post-treatment (not interim) positron emission tomography-computed tomography scan status is highly predictive of outcome in mantle cell lymphoma patients treated with R-HyperCVAD

BACKGROUND:

Although convincing data exist regarding the prognostic utility of positron emission tomographic (PET)‐computed tomographic (CT) imaging in Hodgkin lymphoma and diffuse large B‐cell lymphoma, its prognostic utility both during treatment and immediately after treatment have not been systematically evaluated in a large mantle cell lymphoma (MCL) patient cohort to support its use in clinical practice.

METHODS:

The authors conducted a retrospective cohort study to examine the prognostic utility of PET‐CT imaging in a uniform MCL patient cohort undergoing dose‐intensive chemotherapy (R‐HyCVAD) in the frontline setting. The primary study endpoints were progression‐free survival (PFS) and overall survival (OS). PET‐CT images were centrally reviewed for the purposes of this study using standardized response criteria.

RESULTS:

Fifty‐three patients with advanced stage MCL with PET‐CT data were identified. With median follow‐up of 32 months, 3‐year PFS and OS estimates were 76% (95% confidence interval [CI], 64%‐84%) and 84% (95% CI, 72%‐90%), respectively. Interim PET‐CT status was not associated with PFS (hazard ratio [HR], 0.9; 95% CI, 0.3‐2.7; P = .8) or OS (HR, 0.6; 95% CI, 0.1‐2.9; P = .5). Post‐treatment PET‐CT status was statistically significantly associated with PFS (HR, 5.2; 95% CI, 2.0‐13.6; P = .001) and trended toward significant for OS (HR, 2.8; 95% CI, 0.8‐9.6; P = .07).

CONCLUSIONS:

These data do not support the prognostic utility of PET‐CT in pretreatment and interim treatment settings. A positive PET‐CT after the completion of therapy identifies a patient subset with an inferior PFS and a trend toward inferior OS. Cancer 2012;3565–3570. © 2011 American Cancer Society.     

Etiologic profile of spastic quadriplegia in children

The etiologic profile and possible predictors of etiology in children with spastic quadriplegia were assessed in a consecutive cohort of children with this motor impairment. Medical records from a single pediatric neurology practice over a 14-year interval were retrospectively and systematically reviewed. Variables comprised possible demographic, prenatal, perinatal, and postnatal risk factors. Of the 99 patients included in the study, 39 were premature (<37 weeks gestation). The overall etiologic yield was 83%. The top three diagnoses were hypoxic-ischemic perinatal asphyxia (33%), periventricular leukomalacia (15%), and central nervous system infections (11%). In premature children, the most common diagnoses were periventricular leukomalacia (33%), perinatal asphyxia (26%), and central nervous system infections (15%). In term-born children, the most frequent diagnoses were perinatal asphyxia (37%), metabolic disease (12%), and structural malformation or infection (9% each). Factors predicting the identification of an etiology included male sex (P = 0.05), low birth weight (P = 0.003), prematurity (P = 0.01), perinatal complications (P = 0.002), and neonatal encephalopathy (P = 0.006). The etiologic yield in patients with spastic quadriplegia was 83%, with differing underlying etiologies depending on gestational age. These results should help guide physicians in investigating possible underlying etiologies in patients with spastic quadriplegia.     

A grey zone approach for evaluation of 15 short tandem repeat loci in sibship analysis: a pilot study in Indian subjects

The evaluation of 15 STR loci Applied Biosystems Identifiler kit for sibship determination in Indian subjects is reported. Cumulative sibship indices (CSIs) calculated following standard methods in sibling pairs and non-sibling pairs, showed mean values comparable to other reports. Mean CSI value in sibling group was higher than in corresponding non-sibling group. Moderately high value of CSI in one of the non-sibling pairs and a very low likelihood ratio favoring non-relatedness in a known sibling pair did not allow binary decision about sibship status. To deal with this problem a grey zone approach has been applied to sibship test. It is concluded that the 15 loci STR kit can be reliably used for inferring sibship between pairs of individuals by defining a grey zone of a sibship test as an area of likelihood ratio values where the discriminatory performance is insufficient.     

26-Week dermal oncogenicity study evaluating pure trans-capsaicin in Tg.AC hemizygous mice (FBV/N)

The objective of this study was to assess the oncogenic potential of trans-capsaicin when administered weekly via topical application to the dorsal skin of Tg.AC mice for 26 weeks. Male and female Tg.AC mice (25 mice/sex/group) received dose formulations containing trans-capsaicin dissolved in diethylene glycol monoethyl ether (DGME). The positive control was tetradecanoylphorbol-13-acetate (TPA) dissolved in DGME. Appropriate controls, including a topical lidocaine local anesthetic pretreatment (4%w/w), were maintained. All groups were dosed once weekly, except for the TPA group, which was dosed twice per week. Analysis of the macroscopic observations after the final sacrifice revealed no noteworthy treatment-related findings, with the exception of dermal masses that were randomly dispersed throughout all treatment groups for both males and females. The frequency of dermal masses in the capsaicin-treated groups (at a dose level of up to 102 mg/kg and an application rate of 25.6 mg/cm2/kg/week) was not elevated in comparison to either concurrent vehicle or untreated controls. In contrast, a notable increase in the frequency of dermal masses was observed in the TPA-treated mice compared to both the concurrent vehicle and untreated controls. Dermal application of capsaicin resulted in no increased incidence of preneoplastic or neoplastic skin lesions. In contrast, over half the male and female mice exposed to TPA had multiple skin papillomas; the majority of the TPA-treated animals either died early or was humanely euthanized due to tumor load. Spontaneously occurring neoplasms were not appreciably increased in capsaicin-treated animals. Capsaicin-related non-neoplastic microscopic findings were seen sporadically in both genders and included acanthosis, hyperkeratosis/parakeratosis (primarily females), epidermal crusts, subepidermal fibrosis, epidermal ulcerations/erosions, and chronic-active inflammation. There was no evidence of a dose response in either the incidence or severity of these findings. The lidocaine- (at a dose level of 162 mg/kg and at an application rate of 40.5 mg/cm2/kg/week) and DGME-treated (at a dose level of 4.0 g/kg and at an application rate of 1 g/cm2/kg/week) control groups also did not display any evidence of increase in dermal masses. Based on these results, trans-capsaicin, lidocaine, and DGME should be considered nononcogenic in the Tg.AC mouse dermal model.     

Pharmacokinetics of saquinavir with atazanavir or low-dose ritonavir administered once daily (ASPIRE I) or twice daily (ASPIRE II) in seronegative volunteers

ASPIRE I and II were prospective, 3-way sequential crossover studies in healthy volunteers to compare the safety and pharmacokinetics of saquinavir/ritonavir (SQV/RTV) with saquinavir/atazanavir (SQV/ATV) administered either once daily (QD, ASPIRE I) or twice daily (BID, ASPIRE II). Treatments were separated by 10 days, and pharmacokinetic analyses were performed on days 11, 32, and 53. SQV pharmacokinetics were significantly higher when dosed with RTV compared to ATV (P < .05 for all comparisons). ATV pharmacokinetics were similar within treatment arms. ATV Cmin increased approximately 60%, and Cmax decreased approximately 35% with BID dosing compared with QD dosing. Women had higher exposure for all 3 protease inhibitors (PIs) compared with men after adjusting for weight. Adverse effects were primarily gastrointestinal-related with SQV/RTV and hyperbilirubinemia with SQV/ATV. Although SQV plasma concentrations were higher when coadministered with RTV, a combination of SQV/ATV administered BID may be a viable alternative in HIV-infected, PI-naive subjects intolerant to RTV.     

Influence of acclimation and cross-acclimation of metals on acute Cd toxicity and Cd uptake and distribution in rainbow trout (Oncorhynchus mykiss)

The development of chronic metal toxicity models for fresh water fish is complicated by the physiological adjustments made by the animal during exposure which results in acclimation. This study examines the influence of a pre-exposure to a chronic sublethal waterborne metal on acclimation responses as well as the uptake and distribution of new metal into juvenile rainbow trout. In one series of tests, trout were exposed to either 20 or 60 microg/L Cu, or 150 microg/L Zn for a month in moderately hard water and then cross-acclimation responses to Cd were measured in 96 h LC(50) tests. Cu exposed trout showed a cross-acclimation response but Zn exposed trout did not. Using these results, a detailed examination of Cd uptake and tissue distribution in metal-acclimated trout was done. Trout were exposed to either 75 microg/L Cu or 3 microg/L Cd for 1 month to induce acclimation and subsequently, the uptake and distribution of new Cd was assessed in both Cd- and Cu-acclimated fish using (109)Cd. The pattern of accumulation of new metal was dramatically altered in acclimated fish. For example, in 3 h gill Cd binding experiments, Cd- and Cu-acclimated trout both had a higher capacity to accumulate new Cd but only Cu-acclimated fish showed a higher affinity for Cd compared to unexposed controls. Experiments measuring Cd uptake over 72 h at 3 microgCd/L showed that the Cd uptake rate was lower for Cd-acclimated fish compared to both Cu-acclimated fish and unexposed controls. The results demonstrate the phenomenon of cross-acclimation to Cd and that chronic sublethal exposure to one metal can alter the uptake and tissue distribution of another. Understanding how acclimation influences toxicity and bioaccumulation is important in the context of risk assessment. This study illustrates that knowledge of previous exposure conditions is essential, not only for the metal of concern, but also for other metals as well.     

Targeted inactivation of fh1 causes proliferative renal cyst development and activation of the hypoxia pathway

Germline mutations in the fumarate hydratase (FH) tumor suppressor gene predispose to leiomyomatosis, renal cysts, and renal cell cancer (HLRCC). HLRCC tumors overexpress HIF1alpha and hypoxia pathway genes. We conditionally inactivated mouse Fh1 in the kidney. Fh1 mutants developed multiple clonal renal cysts that overexpressed Hif1alpha and Hif2alpha. Hif targets, such as Glut1 and Vegf, were upregulated. We found that Fh1-deficient murine embryonic stem cells and renal carcinomas from HLRCC showed similar overexpression of HIF and hypoxia pathway components to the mouse cysts. Our data have shown in vivo that pseudohypoxic drive, resulting from HIF1alpha (and HIF2alpha) overexpression, is a direct consequence of Fh1 inactivation. Our mouse may be useful for testing therapeutic interventions that target angiogenesis and HIF-prolyl hydroxylation.