Correlation of mRNA delivery timing and protein expression in lipid-based transfection.

Non-viral gene delivery is constrained by the dwell time that most synthetic nucleic acid nanocarriers spend inside endosomal compartments. In order to overcome this endosomal-release bottleneck, methods are required that measure nanocarrier uptake kinetics and transfection efficiency simultaneously. Here, we employ live-cell imaging on single-cell arrays (LISCA) to study the delivery-time distribution of lipid-based mRNA complexes under varied serum conditions. By fitting a translation-maturation model to hundreds of individual eGFP reporter fluorescence time courses, the protein expression onset times and the expression rates after transfection are determined. Using this approach, we find that delivery timing and protein expression rates are not intrinsically correlated at the single-cell level, even though population-averaged values of both parameters conjointly change as a function of increasing external serum protein fraction. Lipofectamine-mediated delivery showed decreased transfection efficiency and longer delivery times with increasing serum protein concentration. This is in contrast to ionizable lipid nanoparticle (i-LNP)-mediated transfer, which showed increased efficiency and faster uptake in the presence of serum. In conclusion, the interdependences of single-cell expression rates and onset timing provide additional clues on uptake and release mechanisms, which are useful for improving nucleic acid delivery.

Prognostic value of MACC1 and proficient mismatch repair status for recurrence risk prediction in stage II colon cancer patients: the BIOGRID studies.

BACKGROUND: We assessed the novel MACC1 gene to further stratify stage II colon cancer patients with proficient mismatch repair (pMMR). PATIENTS AND METHODS: Four cohorts with 596 patients were analyzed: Charité 1 discovery cohort was assayed for MACC1 mRNA expression and MMR in cryo-preserved tumors. Charité 2 comparison cohort was used to translate MACC1 qRT-PCR analyses to FFPE samples. In the BIOGRID 1 training cohort MACC1 mRNA levels were related to MACC1 protein levels from immunohistochemistry in FFPE sections; also analyzed for MMR. Chemotherapy-naïve pMMR patients were stratified by MACC1 mRNA and protein expression to establish risk groups based on recurrence-free survival (RFS). Risk stratification from BIOGRID 1 was confirmed in the BIOGRID 2 validation cohort. Pooled BIOGRID datasets produced a best effect-size estimate. RESULTS: In BIOGRID 1, using qRT-PCR and immunohistochemistry for MACC1 detection, pMMR/MACC1-low patients had a lower recurrence probability versus pMMR/MACC1-high patients (5-year RFS of 92% and 67% versus 100% and 68%, respectively). In BIOGRID 2, longer RFS was confirmed for pMMR/MACC1-low versus pMMR/MACC1-high patients (5-year RFS of 100% versus 90%, respectively). In the pooled dataset, 6.5% of patients were pMMR/MACC1-low with no disease recurrence, resulting in a 17% higher 5-year RFS [95% confidence interval (CI) (12.6%-21.3%)] versus pMMR/MACC1-high patients (P = 0.037). Outcomes were similar for pMMR/MACC1-low and deficient MMR (dMMR) patients (5-year RFS of 100% and 96%, respectively). CONCLUSIONS: MACC1 expression stratifies colon cancer patients with unfavorable pMMR status. Stage II colon cancer patients with pMMR/MACC1-low tumors have a similar favorable prognosis to those with dMMR with potential implications for the role of adjuvant therapy.

Noise thermometry at ultra-low temperatures.

The options for primary thermometry at ultra-low temperatures are rather limited. In practice, most laboratories are using (195)Pt NMR thermometers in the microkelvin range. In recent years, current sensing direct current superconducting quantum interference devices (DC-SQUIDs) have enabled the use of noise thermometry in this temperature range. Such devices have also demonstrated the potential for primary thermometry. One major advantage of noise thermometry is the fact that no driving current is needed to operate the device and thus the heat dissipation within the thermometer can be reduced to a minimum. Ultimately, the intrinsic power dissipation is given by the negligible back action of the readout SQUID. For thermometry in low-temperature experiments, current noise thermometers and magnetic flux fluctuation thermometers have proved to be most suitable. To make use of such thermometers at ultra-low temperatures, we have developed a cross-correlation technique that reduces the amplifier noise contribution to a negligible value. For this, the magnetic flux fluctuations caused by the Brownian motion of the electrons in our noise source are measured inductively by two DC-SQUID magnetometers simultaneously and the signals from these two channels are cross-correlated. Experimentally, we have characterized a thermometer made of a cold-worked high-purity copper cylinder with a diameter of 5 mm and a length of 20 mm for temperatures between 42 µK and 0.8 K. For a given temperature, a measuring time below 1 min is sufficient to reach a precision of better than 1%. The extremely low power dissipation in the thermometer allows continuous operation without heating effects.

Nuclear magnetic resonance and dielectric noise study of spectral densities and correlation functions in the glass forming monoalcohol 2-ethyl-1-hexanol.

The spectral densities related to various relaxation processes of the glass former 2-ethyl-1-hexanol (2E1H), a monohydroxy alcohol, are probed using several nuclear magnetic resonance (NMR) experiments as well as via dielectric noise spectroscopy (DNS). On the basis of the spectral density relating to voltage fluctuations, i.e., without the application of external electrical fields, DNS enables the detection of the structural relaxation and of the prominent, about two decades slower Debye process. The NMR-detected spectral density, sensitive to the orientational fluctuations of the hydroxyl deuteron, also reveals dynamics slower than the structural relaxation, but not as slow as the Debye process. Rotational and translational correlation functions of 2E1H are probed using stimulated-echo NMR techniques which could only resolve the structural dynamics or faster processes. The experimental results are discussed with reference to models that were suggested to describe the dynamics in supercooled alcohols.

Fluctuation-dissipation theorem in liquid and glassy glycerol: frequency-dependent dielectric permittivity and dielectric polarization fluctuation measurements.

We show frequency-dependent dielectric permittivity and dielectric polarization fluctuation measurements of liquid and glassy glycerol. This allows a direct comparison of both quantities determined independently. After cooling the glycerol sample to 179 K with a cooling rate of 0.85 K/min we studied the aging time dependence of the dielectric permittivity and the polarization fluctuations using the identical glycerol sample. A cross-correlation technique allows measurements of noise levels below the amplifier noise. In the frequency range between 0.3 and 300 Hz we find the measured data to be in agreement with the fluctuation-dissipation theorem for the liquid and glassy state not depending on the aging time.

Beating of coupled ultraviolet light modes in zinc oxide nanoresonators.

Periodic spatial intensity modulations of near-band-gap luminescence light at 3.36 eV photon energy are reported in nanometer-sized resonators of single zinc oxide nanorods. These modulations are observed when the luminescence light, due to excitonic recombination, is excited by a focused low-energy electron beam scanned along the rods. The acceleration energy of only 2 keV allows for a spatial resolution of a few ten nanometers. Numerical calculations based on the theory of dielectric cylindrical waveguides are performed identifying the intensity modulations as beating patterns of coupled TE01 and TM01 modes in the nanoresonator.

Her-2/neu expression in breast cancer--A comparison of different diagnostic methods.

BACKGROUND: Determination of Her-2/neu overexpression in breast cancer has previously been shown to be of prognostic significance. In this study, Her-2/neu expression in breast cancer was characterised by real-time PCR (RLT-PCR) based LightCycler-HER-2/neu DNA Quantification with immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). MATERIAL AND METHODS: Fifteen specimens of invasive breast cancer - whole tissue sections as well as microdissected tumour cells - were subjected to RLT-PCR. Additionally, IHC and FISH were performed. RESULTS: Her-2/neu overexpression was detected by FISH and by real-time PCR in the same tumours. In contrast, IHC revealed discordant results. CONCLUSION: Determination of Her-2/neu amplification by real-time PCR is a sensitive and specific method with some advantages over FISH. This method is simple and reliable and has the potential of categorizing those tumours with borderline Her-2/neu overexpression as determined by IHC.

Effect of pressure on the secondary relaxation in a simple glass former.

We have studied dielectric spectra of the glass-forming liquid metafluoroaniline under hydrostatic pressure up to 700 MPa. Its glass transition pressure p(g) increases approximately linearly with temperature. Above p(g)(T), a well pronounced secondary relaxation, the Johari beta peak, is observed showing activated behavior. The activation energy rises proportionally to pressure and, consequently, proportionally to the glass transition temperature T(g)(p). The activation volume is independent of temperature but exhibits different values for pressures higher and lower than the pressure where the liquid left the ergodic regime. The activation volumes are about 1/10 and 1/6 of the molecular volume of fluoroaniline, respectively, suggesting that there are two different species of clusters.

Susceptibility testing of macrolide and lincosamide antibiotics according to DIN guidelines. Deutsches Institut für Normung.

The in-vitro activity of azithromycin, clarithromycin, erythromycin, josamycin, midekamycin, roxithromycin and clindamycin against 674 Gram-negative and Gram-positive clinical isolates, including methicillin-resistant Staphylococcus aureus, was determined by agar dilution, microdilution and agar diffusion with Mueller-Hinton medium according to the Deutsches Institut für Normung (DIN) 58940 guidelines. The results obtained by regression analysis and the error-rate-bounded method of Metzler-DeHaan indicate that common interpretative criteria (breakpoints) for test discs may be assigned to susceptible/resistant Gram-positive strains for all antibiotics tested. The following tentative DIN values are suggested for 15 microg macrolide discs: for susceptible Gram-positive and Gram-negative strains, an inhibition zone diameter (IZD) of > or = 26 mm at a corresponding MIC of < or = 2 mg/L; for resistant Gram-positive strains, an IZD of < or = 21 mm; for resistant Gram-negative strains, an IZD of < or = 19 mm at a corresponding MIC of > or = 8 mg/L. For Haemophilus influenzae only, breakpoints for azithromycin are suggested with IZDs of > or = 21 mm for susceptible and < or = 18 mm for resistant.

[Differential indications for non-opioids for postoperative analgesia III. Analgesic effect of perioperative administration of metamizole plus diclofenac after spinal anesthesia]. / Untersuchungen zum differenzierten Einsatz von Nichtopioiden zur postoperativen Analgesie III. Analgetischer Effekt einer perioperativen Gabe von Metamizol plus Dicllofenac nach Spinalanästhesien.

PURPOSE: In a previous study we investigated the analgesic efficacy of a combination of metamizol plus diclofenac after general anaesthesia. After minor orthopaedic surgery postoperative opioid requirements were reduced by 73% during the first 24 h after surgery. In the present study, we have investigated the efficacy of this analgesic combination after minor orthopaedic operations performed in spinal anaesthesia. METHODS: Seventy four patients, scheduled for minor orthopaedic surgery, participated in this double-blind, randomised, placebo-controlled study. The setting was comparable to our previous study. Before induction of spinal anaesthesia, verum-treated patients received a diclofenac suppository (100 mg), and metamizol (1 g/100 ml NaCl 0.9% intravenously over 15 min). These infusions were repeated at 6 h and 12 h. In addition to the third infusion, the patients received a further diclofenac suppository (100 mg). Cumulated doses of buprenorphine (PCA, patient-controlled analgesia), pain scores (0-10), blood pressure, heart rate and side effects were recorded during the first 6 h and again at 24 h. RESULTS: After spinal anaesthesia had subsided, all patients required increasing doses of buprenorphine. Verum-treated patients required significantly lower doses during the first 24 h after surgery (median -29%). CONCLUSIONS: The combination of metamizol and diclofenac causes a clinically relevant reduction in opioid requirements after minor orthopaedic surgery in spinal anaesthesia.

[Susceptibility testing of yeasts against fluconazole: comparison of the Etest method with microdilution and agar dilution]. / Empfindlichkeitsprüfung von Hefen gegenüber Fluconazol: Vergleich der Etest-Methode mit der Mikrodilution und der Agardilution.

In bacteriology, the Etest has a broad field of application in bacteriology and is recently available for the antimycotics fluconazole and itraconazole. By means of the presence of gradient concentrations of the active substance on the carrier material, it is possible to obtain reproducible MICs of the antimycotic substances. The results of susceptibility testing of 326 clinical yeast isolates with the Etest were compared to those MICs obtained by microdilution and agar dilution. A 100% concordance of the MIC markers (mode-, MIC50- and MIC90-value, standard deviation of the mean log2-MIC-dilution steps) was given when compared by a +/- 1 MIC-dilution step range with microdilution and by +/- 2 MIC-dilution steps with agar dilution; species dependent all strains were within 2 x standard deviation of the individual MIC-mean of the species. By comparison of the individual MIC-values maximum differences of +/- 6 MIC-dilution steps were obtained, where 70% of all results were within +/- 2 MIC-dilution steps, and more than 92% of all strains were within +/- 3 MIC-dilution steps. The Pearson's correlation coefficients show a good agreement of the Etest with microdilution (r = 0.92) resp., agar dilution (r = 0.88) demonstrate, however, clearly insufficient correlations (r < 0.65) to the reference methods, for species with difficult to read Etest inhibition zones (e.g., Candida glabrata, Candida krusei, Candida parapsilosis). The differences between the proposed test methods recommended by the NCCLS and the working group "Clinical Mycology" of the German Speaking Mycological Society (AG-KMYK) are tabled.

An evaluation of seven methods of testing in vitro susceptibility of clinical yeast isolates to fluconazole.

Four commercially available in vitro test systems (Candifast, E-test, Mycototal and spiral-gradient end point method), agar diffusion with 25-micrograms fluconazole paper test discs and 15-micrograms test tablets and agar dilution were compared with the microbroth dilution method for fluconazole susceptibility testing of 145 clinical isolates. In addition, the culture media provided or recommended by the manufacturers of the test systems were compared with high-resolution (HR) antifungal test medium. With all currently available culture media, growth problems (inhibition or delayed growth of the clinical isolates) occurred with solid or semisolid media. With minor improvements, HR medium demonstrated the most reproducible and comparable results (supplementation with asparagine and deletion of sodium hydrogen carbonate). The best correlation with microdilution was obtained by the agar dilution method (> 95% concordance) followed by the spiral-gradient end point method (85%), Candifast (83%), Mycototal (81%) and the E-test (78%). Regression analysis demonstrated good correlation between agar diffusion and micro-/agar dilution (r > 0.9).

[Comparison of seven methods of in vitro susceptibility testing of clinical yeast isolates against fluconazole]. / Ein Vergleich von sieben Methoden zur In-vitro-Empfindlichkeitsprüfung von klinischen Hefe-Isolaten gegenüber Fluconazol.

Four commercially available in vitro test systems (Candifast, E-test, Mycototal, Spiral-Gradient Endpoint Method), agardiffusion with 25 micrograms fluconazole paper test discs and 15 micrograms test tablets, and agardilution were compared to the microbroth dilution method by fluconazole susceptibility testing of 145 clinical isolates. In addition, the culture media provided or recommended by the manufacturers of the test systems were compared to the high resolution (HR) antifungal test medium. With all currently available culture media growth problems (inhibition or delayed growth of the clinical isolates) occurred with solid or semi-solid media. With minor improvements, HR medium demonstrated the most reproducible and comparable results (supplementation with asparagine and deletion of sodium hydrogen carbonate). Best correlation to microdilution was obtained by the agardilution method > (95% concordance) followed by the spiral gradient endpoint method (85%), Candifast (83%), Mycototal (81%) and the E-test (78%). Regression analysis demonstrated good correlation between agardiffusion and micro-/agardilution(r > 0.9).

Behaviour problems and developmental status of 3-month-old infants in relation to organic and psychosocial risks.

In a prospective longitudinal study starting at birth 384 infants and their families were investigated. The subjects were distributed across the nine cells of a two-factorial design (3 x 3), factor 1 representing the degree of organic, factor 2 the degree of psychosocial risk. At age 3 months there was a significant influence of organic risk factors on all developmental parameters. Psychosocial risks were only relevant for cognitive development and behaviour problems. Organic high-risk children who adapted quickly after birth showed an especially good development status.