TAT Peptide-Conjugated Magnetic PLA-PEG Nanocapsules for the Targeted Delivery of Paclitaxel: <Emphasis Type="Italic">In Vitro</Emphasis> and Cell Studies

Paclitaxel (PTX) and organophilic iron oxide nanocrystals of 7 nm average size were co-encapsulated in the oily core of poly(lactide)-poly(ethyleneglycol) (PLA-PEG) nanocapsules in order to develop magnetically responsive nanocarriers of PTX. The nanocapsules were prepared by a solvent displacement technique and exhibited satisfactory drug and iron oxide loading efficiency, high colloidal stability, and sustained drug release properties. Drug release also proved responsive to an alternating magnetic field. Magnetophoresis experiments showed that the magnetic responsiveness of the nanocapsules depended on their SPION content. The PTX-loaded nanocapsules exhibited comparable to free PTX cytotoxicity against the A549 lung cancer cell line at 24 h of incubation but higher cytotoxicity than free drug at 48 h of incubation. The conjugation of a cysteine-modified TAT peptide (HCys-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH₂) on the surface of the nanocapsules resulted to highly increased uptake of nanocapsules by cancer cells, as well as to profound improvement of their cytotoxicity against the cancer cells. The results obtained justify further investigation of the prospects of these multifunctional PLA-PEG nanocapsules as a targeted delivery system of paclitaxel.     

Function and quality of kidneys after cold storage, machine perfusion, or retrograde oxygen persufflation: Results from a porcine autotransplantation model

Objectives

Especially for preservation of marginal donor organs, machine perfusion (MP) and retrograde oxygen persufflation (ROP) are alternatives to cold storage (CS). Using a porcine kidney autotransplantation model we compared metabolic and morphologic effects of CS, ROP, and MP on kidneys exposed to warm ischemia.

Methods

Kidneys of 21 pigs were exposed in situ to warm ischemia for 60 min. The kidneys were randomly allocated to three experimental groups, each receiving a 4-h treatment of either cold storage, machine perfusion, or retrograde oxygen persufflation. Tissue samples were examined for malondialdehyde and histological changes. Daily blood samples were examined for creatinine levels.

Results

Seven days after transplantation, the plasma creatinine levels in the CS and MP groups were still significantly elevated above the baseline values. In the ROP group, all animals exhibited nearly normal creatinine levels. Malondialdehyde, an indicator of lipidperoxidation, was dramatically increased in the machine perfused kidneys on day 7, whereas the malondialdehyde levels in the other two groups were near normal values. The MP kidneys exhibited the most striking histological changes.

Conclusion

Though MP has been well introduced in organ transplantation, in our opinion, it must still be optimized and standardized. It is necessary to clarify questions such as whether there is a need for oxygenation during perfusion, the length of perfusion, the impact of pressure, and the effects of additional scavengers. The results of the present study suggest the reconsideration of the ROP-technique for the preservation of predamaged donor grafts especially of NHBD and further studies, comparing MP and ROP upon long term preservation are strongly encouraged.     

Impact of heat waves on mortality in Croatia

The aim of this work was to determine the criteria for heat loads associated with an increase in mortality in different climatic regions of Croatia. The relationship between heat stress and mortality was analysed for the period 1983–2008. The input series is excess mortality defined as the deviations of mortality from expected values determined by means of a Gaussian filter of 183 days. The assessment of the thermal environment was performed by means of physiologically equivalent temperature (PET). The curve depicting the relationship between mortality and temperature has a U shape, with increased mortality in both the cold and warm parts of the scale but more pronounced in the warm part. The threshold temperature for increased mortality was determined using a scatter plot and fitting data by means of moving average of mortality; the latter is defined as the temperature at which excess mortality becomes significant. The values are higher in the continental part of Croatia than at the coast due to the refreshing influence of the sea during the day. The same analysis on a monthly basis shows that at the beginning of the warm season increased mortality occurs at a lower temperature compared with later on in the summer, and the difference is up to 15 °C between August and April. The increase in mortality is highest during the first 3–5 days and after that it decreases and falls below the expected value. Long-lasting heat waves present an increased risk, but in very long heat waves the increase in mortality is reduced due to mortality displacement.     

Head-to-head comparison of BNP and IL-6 as markers of clinical and experimental heart failure: Superiority of BNP

Activation of BNP and IL-6 are hallmarks of left ventricular (LV) dysfunction and congestive heart failure (CHF). To assess the relative activation of BNP and IL-6 in clinical and experimental heart failure, we performed a human study in which plasma N-terminal proBNP (NT-proBNP) and IL-6 were measured in a large group of patients in the chronic phase after myocardial infarction (MI) and an animal study in which LV gene expression of BNP and IL-6 was assessed in rapid ventricular pacing-induced heart failure. In the human study, NT-proBNP and IL-6 were measured by non-extracted, enzyme-linked immunoassay in 845 subjects (n=468 outpatients after MI, MONICA MI register Augsburg; and 377 siblings without MI, control). NT-proBNP (295+/-23pg/mL vs. CTRL 84+/-8, P<0.05) and IL-6 (2.7+/-0.1pg/mL vs. CTRL 2.1+/-0.1, P<0.05) were both elevated in subjects with MI. These increases were particularly pronounced in the presence of concomitant CHF (both P<0.01 vs. CTRL) and LV dysfunction (EF<45%, both P<0.05 vs. CTRL). However, NT-proBNP was significantly correlated with several cardiac structural and functional parameters (EF, LVMI, history of MI, CHF symptoms; all P<0.05) upon regression analysis whereas IL-6 was only correlated with history of MI (P<0.001). Accordingly, MI subjects with symptomatic LV dysfunction were detected by NT-proBNP with a greater sensitivity, specificity, and ROC-area (85%, 88%, and 0.87, respectively) as compared to IL-6 (69%, 53%, and 0.67, respectively). In the animal study, IL-6 and BNP expression were both significantly elevated in CHF (both P<0.05) but with a much greater absolute activation of BNP. In addition, BNP mRNA expression displayed a stronger inverse correlation with LV function (r=-0.74; P<0.001) than IL-6 (r=-0.53; P=0.001) and was a markedly more sensitive and specific molecular marker of LV dysfunction (sensitivity 91%, specificity 100%, ROC-area 0.94) than IL-6 (sensitivity 74%, specificity 83%, ROC-area 0.87). Our animal study provides evidence that IL-6 expression is activated in heart failure but to a significantly lesser degree than that of BNP. Both the stronger expression of BNP and the better correlation with LV function provide the molecular basis for a diagnostic superiority of NT-proBNP in clinical LV dysfunction and heart failure.     

Molecular Phylogeny of Metazoan Intermediate Filament Proteins

We have cloned cytoplasmic intermediate filament (IF) proteins from a large number of invertebrate phyla using cDNA probes, the monoclonal antibody IFA, peptide sequence information, and various RT-PCR procedures. Novel IF protein sequences reported here include the urochordata and nine protostomic phyla, i.e., Annelida, Brachiopoda, Chaetognatha, Echiura, Nematomorpha, Nemertea, Platyhelminthes, Phoronida, and Sipuncula. Taken together with the wealth of data on IF proteins of vertebrates and the results on IF proteins of Cephalochordata, Mollusca, Annelida, and Nematoda, two IF prototypes emerge. The L-type, which includes 35 sequences from 11 protostomic phyla, shares with the nuclear lamins the long version of the coil 1b subdomain and, in most cases, a homology segment of some 120 residues in the carboxyterminal tail domain. The S-type, which includes all four subfamilies (types I to IV) of vertebrate IF proteins, lacks 42 residues in the coil 1b subdomain and the carboxyterminal lamin homology segment. Since IF proteins from all three phyla of the chordates have the 42-residue deletion, this deletion arose in a progenitor prior to the divergence of the chordates into the urochordate, cephalochordate, and vertebrate lineages, possibly already at the origin of the deuterostomic branch. Four phyla recently placed into the protostomia on grounds of their 18S rDNA sequences (Brachiopoda, Nemertea, Phoronida, and Platyhelminthes) show IF proteins of the L-type and fit by sequence identity criteria into the lophotrochozoic branch of the protostomia. Received: 2 April 1998 / Accepted: 19 June 1998     

Abnormal motor activity during anaesthesia in a dog: a case report

Seizures or convulsions that occur during anaesthesia in veterinary patients are infrequently reported in the literature. Consequently, the incidence of such events is unknown. Several drugs commonly used in clinical veterinary anaesthesia have been shown to induce epileptiform activity in both human clinical patients and experimental candidates. The present case report describes convulsions in a four-year old male Bernese mountain dog during maintenance of anaesthesia with isoflurane after premedication with acepromazine and methadone followed by co-induction with propofol and ketamine. The dog had no history of previous convulsions. The use of several sedative and anaesthetic drugs makes it difficult to find one single causative pharmaceutical.     

An “orientation sphere” visualization for examining animal head movements

1. Animal behavior is elicited, in part, in response to external conditions, but understanding how animals perceive the environment and make the decisions that bring about these behavioral responses is challenging.
2. Animal heads often move during specific behaviors and, additionally, typically have sensory systems (notably vision, smell, and hearing) sampling in defined arcs (normally to the front of their heads). As such, head‐mounted electronic sensors consisting of accelerometers and magnetometers, which can be used to determine the movement and directionality of animal heads (where head “movement” is defined here as changes in heading [azimuth] and/or pitch [elevation angle]), can potentially provide information both on behaviors in general and also clarify which parts of the environment the animals might be prioritizing (“environmental framing”).
3. We propose a new approach to visualize the data of such head‐mounted tags that combines the instantaneous outputs of head heading and pitch in a single intuitive spherical plot. This sphere has magnetic heading denoted by “longitude” position and head pitch by “latitude” on this “orientation sphere” (O‐sphere).
4. We construct the O‐sphere for the head rotations of a number of vertebrates with contrasting body shape and ecology (oryx, sheep, tortoises, and turtles), illustrating various behaviors, including foraging, walking, and environmental scanning. We also propose correcting head orientations for body orientations to highlight specific heading‐independent head rotation, and propose the derivation of O‐sphere‐metrics, such as angular speed across the sphere. This should help identify the functions of various head behaviors.
5. Visualizations of the O‐sphere provide an intuitive representation of animal behavior manifest via head orientation and rotation. This has ramifications for quantifying and understanding behaviors ranging from navigation through vigilance to feeding and, when used in tandem with body movement, should provide an important link between perception of the environment and response to it in free‐ranging animals.

     

Paleoecology of Upper Eifelian and Lower Givetian coral limestones in the northwestern Sauerland (Devonian; Rhenish Massif)

Summary The prevailing sandy/silty lower part of the Middle Devonian in the northwestern Sauerland includes two coral limestone horizons, which contain a rich fauna of corals, stromatoporoids, and calcareous algae. The Ihmert-Formation is subdvided into three parts. The older coral limestone horizon is the Grünewiese-Member of the Ihmert-Formation (uppermost Eifelian), the younger is in the Bredenbruch-Member of the Unterhonsel-Formation (lower Lower Givetian). Conclusions about the environmental constraints are drawn from the sedimentology and the fossil content of the coral limestones. Predominant biostromes are built between storm wave base and normal wave base. Only the few bioherms grew above the normal wave base. These coral limestones were deposited in a tropical or subtropical normal marine environment in the shallow euphotic zone. Among the reef-builders epoecism is very frequent, and until now this phenomenon has not been investigated in detail. Fragile rugose and tabulate corals lived as commensals with stromatoporoids. Some other aspects of paleoecology are concisely presented.     

C and N allocation in soil under ryegrass and alfalfa estimated by 13C and 15N labelling

Background and Aims

Below-ground translocated carbon (C) released as rhizodeposits is an important driver for microbial mobilization of nitrogen (N) for plants. We investigated how a limited substrate supply due to reduced photoassimilation alters the allocation of recently assimilated C in plant and soil pools under legume and non-legume species.

Methods

A non-legume (Lolium perenne) and a legume (Medicago sativa) were labelled with ¹⁵N before the plants were clipped or shaded, and labelled twice with ¹³CO₂ thereafter. Ten days after clipping and shading, the ¹⁵N and ¹³C in shoots, roots, soil, dissolved organic nitrogen (DON) and carbon (DOC) and in microbial biomass, as well as the ¹³C in soil CO₂ were analyzed.

Results

After clipping, about 50 % more ¹³C was allocated to regrowing shoots, resulting in a lower translocation to roots compared to the unclipped control. Clipping also reduced the total soil CO₂ efflux under both species and the ¹³C recovery of soil CO₂ under L. perenne. The ¹⁵N recovery increased in the shoots of M. sativa after clipping, because storage compounds were remobilized from the roots and/or the N uptake from the soil increased. After shading, the assimilated ¹³C was preferentially retained in the shoots of both species. This caused a decreased ¹³C recovery in the roots of M. sativa. Similarly, the total soil CO₂ efflux under M. sativa decreased more than 50 % after shading. The ¹⁵N recovery in plant and soil pools showed that shading has no effect on the N uptake and N remobilization for L. perenne, but, the ¹⁵N recovery increased in the shoot of M. sativa.

Conclusions

The experiment showed that the dominating effect on C and N allocation after clipping is the need of C and N for shoot regrowth, whereas the dominating effect after shading is the reduced substrate supply for growth and respiration. Only slight differences could be observed between L. perenne and M. sativa in the C and N distribution after clipping or shading.