Design of potent inhibitors of HIV-1 entry from the gp41 N-peptide region

The HIV-1 gp41 envelope glycoprotein promotes fusion of the virus and cell membranes through the formation of a trimer-of-hairpins structure, in which the amino- and carboxyl-terminal regions of the gp41 ectodomain are brought together. Synthetic peptides derived from these two regions (called N and C peptides, respectively) inhibit HIV-1 entry. In contrast to C peptides, which inhibit in the nanomolar range, N peptides are weak inhibitors with IC(50) values in the micromolar range. To test the hypothesis that the weak inhibition of N peptides results from their tendency to aggregate, we have constructed chimeric variants of the N-peptide region of gp41 in which soluble trimeric coiled coils are fused to portions of the gp41 N peptide. These molecules, which present the N peptide in a trimeric coiled-coil conformation, are remarkably more potent inhibitors than the N peptides themselves and likely target the carboxyl-terminal region of the gp41 ectodomain. The best inhibitors described here inhibit HIV-1 entry at nanomolar concentrations.     

Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H- vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.     

Interplay between toothbrush stiffness and dentifrice abrasivity on the development of non-carious cervical lesions

Objective

This study investigated the effect of toothbrush stiffness and dentifrice slurry abrasivity on the development and progression of simulated non-carious cervical lesions (NCCLs).

Materials and methods

Human maxillary premolars were allocated to 12 groups generated by the association between toothbrushes, soft, medium, and hard stiffness, and simulated dentifrice slurries, lower, medium, and higher; deionized water (DI) served as negative control. Teeth were mounted on acrylic blocks, and their root surfaces partially covered with acrylic resin to simulate gingiva, leaving a 2-mm area apical to the cemento-enamel junction exposed to toothbrushing. Specimens were brushed with the test slurries for 35,000 and 65,000 double strokes. Impressions taken at baseline and after both brushing periods were scanned by a 3D optical profilometer. Dentin volume loss (mm³) was calculated by image subtraction. Data were analyzed using three-way ANOVA and Fisher’s PLSD tests.

Results

All toothbrushes caused higher volume loss when associated to higher abrasive slurry, compared to medium- and lower-abrasive slurries. Medium caused more volume loss than lower-abrasive slurry, which led to more volume loss than DI. Hard and medium toothbrushes were not different when used with medium- or higher-abrasive slurries. There were no differences among toothbrushes when used with DI and lower-abrasive slurry. Overall, 35,000 brushing strokes resulted in significantly less volume loss than 65,000.

Conclusions

Toothbrush stiffness was an important factor on NCCL development, especially when brushing with medium- and higher-abrasive slurries.

Clinical relevance

Medium and hard toothbrushes associated with medium- and high-abrasive toothpastes can yield more severe NCCLs.

     

Physical activity behaviour in children and adolescents before,during and after cancer treatment

Purpose

Childhood cancer survivors show reduced physical activity (PA) levels which may considerably impact child development, quality of life, social participation and sequelae such as functional and cardiovascular health. This study aims to evaluate different aspects of PA behaviour in patients with childhood cancer (PaC) before (bT), during (dT) and after (aT) cancer treatment.

Methods

In this cross-sectional, multicentre study, 114 PaC and 37 healthy controls between 4 and 20 years of age were enrolled. PA behaviour was assessed using an adapted questionnaire which included items asking about PA level, PA intensity and domains of PA.

Results

Patients reported lower PA levels and less minutes of PA at moderate-intensity dT than aT and bT (P?≤?0.05). Healthy controls reported higher PA levels than patients aT (P?≤?0.05). At school, 41.7% of PaC did not participate in physical education aT or bT. Lastly, 45.6% of PaC who were engaged in sport club activities bT did no more participate in sport club activities aT.

Conclusion

Patients reported different PA behaviours dT and aT than bT. Therefore, monitoring of PA should be considered to increase PA levels in PaC. Future studies also need to examine how PA behaviour can be influenced in a positive way in PaC.

     

Bacterial colonization of microbial biofilms in oral squamous cell carcinoma

Objectives

The purpose of this prospective clinical study was to identify the bacterial spectra on the surface of oral squamous cell carcinomas (OSCC) in comparison to oral mucosa of patients with a higher risk to emerge an OSCC and a control group to determine their susceptibility to various common antibiotics.

Material and methods

Swabs from 90 patients, 30 patients of each group, were cultured on media for aerobes and anaerobes and tested with agar diffusion and Etest.

Results

The predominant pathogens of the normal healthy oral mucosa were aerobes. The ratio between aerobes and anaerobes was 2:1, balanced in risk patients and inverted in the OSCC group. Altogether, 1,006 isolates were cultured. The most frequent strains were 47 viridans streptococci, 30 Staphylococcus species, 14 Enterococcus faecalis, 36 Neisseria species, 14 Escherichia coli, and 23 other aerobes, 66 Peptostreptococcus species, 39 Fusobacterium species, and 34 Prevotella species. The resistance rates in the OSCC group were penicillin 40 %, ampicillin 57 %, doxycycline 23 %, clindamycin 47 %, and amoxicillin/clavulanic acid 20 %, but up to 100 % of pathogens were susceptible to azithromycin, telithromycin, levofloxacin, and moxifloxacin.

Conclusion

Gram-negative anaerobes play a decisive role in the development of postoperative infections in patients with OSCC. This tumor special type of colonization does not agree with the normal flora of the oral cavity. Clinical relevance: Biofilms on OSCC surfaces provide an important reservoir for anaerobic bacteria. As a consequence, a proposal for an antibiotic prophylactic regime should be given.     

Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80–540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.