Femoral neck fracture after removal of the standard gamma interlocking nail: a cadaveric study to determine factors influencing the biomechanical properties of the proximal femur

We retrospectively analyzed 1334 patients who were implanted standard gamma interlocking nails^® (SGN) to stabilize trochanteric femoral fractures over the years 1992–1998. Reoperation to remove the nails was performed in 37 patients, in 9 of them purely because of pain. Three out of these 9 patients with removed SGN suffered femoral neck fractures in the early postoperative course after having been mobilized to full weight-bearing capacity. This complication was not observed with other implant systems and, considering the notoriously high complication rate of femoral neck fractures, severely reduces the value of the SGN concept per se. These findings in combination with other known shortcomings of SGNs prompted us to conduct an experimental study on the fracture force of excavated femurs addressing the hypothesis that the specific design of the SGN is responsible for the occurrence of fatigue fractures of the femoral neck. Eighteen matched pairs of fresh human cadaveric proximal femurs, which were treated by insertion and removal of (i) SGNs or (ii) dynamic hip screws^® (DHS) or (iii) by excavation in the absence of an implant, were subjected to incremental loading cycles and compared to the untreated contralateral femurs. Overall, the fracture force was found to be significantly lower among the treated than among the untreated bones. However, the fracture force required after removal of the DHS system was still significantly higher than for SGN or excavation alone. In this way, our findings demonstrate that removing relatively big implants such as SGN can cause serious complications such as femoral neck fractures. We therefore recommend to leave this type of device in place even after fracture healing except in cases of deep and chronic infection.     

Adventitious root induction in Ophiorrhiza prostrata: a tool for the production of camptothecin (an anticancer drug) and rapid propagation

Roots of Ophiorrhiza prostrata D. Don serve as a rich source of camptothecin (CPT), an anticancer drug. Because of the large-scale collection of its roots, the plant has become a threatened species. The present study accomplishes the induction of adventitious roots as a means for the production of CPT as well as for the large-scale propagation of this anticancer drug plant using leaf and internode explants. The biomass yield and CPT content of adventitious roots induced from different explants were compared to roots developed on ex vitro rooted stem cuttings. Adventitious roots were produced on half-strength Murashige and Skoog (MS) medium supplemented with 10.74 μM α-naphthaleneacetic acid and 2.32 μM kinetin at mean fresh weights of 0.753, 0.739 and 0.748 g roots from leaf, internode and shoot, respectively. CPT yield from in vitro derived roots after 50, 80 and 120 days of incubation (0.028, 0.06 and 0.1% dry weight, respectively) was not significantly different from those harvested at the same age from ex vitro rooted (0.03, 0.06 and 0.13%, respectively) stem cuttings. CPT from subcultured roots derived from solid (0.08%) medium was lower than from suspension culture medium (0.12%). Subsequent cultures of the adventitious roots showed a stable production of CPT (0.16%). The yield of CPT from 360-day-old plant-derived roots was 0.19%. Elicitation using methyl jasmonate and acetyl salicylic acid exhibited no enhancement in CPT yield. In vitro propagation through direct shoot regeneration was achieved from the adventitious roots upon transfer to MS medium with 8.87 μM N ⁶-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) with a mean of 21.2 shoots per culture in 50 days. The shoots upon subculture on medium having the same level of BA and IBA underwent rapid proliferation. The shoots transferred to field conditions after in vitro rooting exhibited 95% survival. Adventitious root induction, from leaf and internode explants, enables the feasible production of CPT as well as the large-scale rapid propagation of this species which can safeguard it from extinction.     

Modelling hydrolysis of leaf litter by digestive enzymes of the snail Melanopsis praemorsa: combination of response surface methodology and in vitro assays

The objective of the present study was to test the application of an in vitro assay simulating the digestive hydrolysis of leaf litter by the freshwater snail M. praemorsa, as well as to determine the possible influence of different factors in the efficiency of such process to release biologically available C and N under the forms of reducing sugars and amino acids from two different substrates. A novel approach to construct a model explaining the effect of three main factors (temperature, total reaction time and enzyme:substrate ratio) in the digestive hydrolysis of cellulose and protein present in leaf litter of different nutritive value is used. The methodology combines a factorial design based in the response surface methodology (RSM) and in vitro digestibility assays adapted to the physiology of both plant substrates used (alder and poplar leaves). The model revealed a different influence of the factors in the hydrolysis of two plant substrates, poplar and alder leaves and the main effect was produced by the time available for hydrolysis. A compensation response based in a longer gut retention time for the lower quality substrate was observed in the feeding assays. The use of in vitro assays and RSM provides a useful insight on the effect of factors and mechanisms underlying the observed differences in nutritional value of leaf litter for an aquatic invertebrate, being such differences linked to the whole bioavailability of carbon and nitrogen in headwater streams.     

Comet assay: a reliable tool for the assessment of DNA damage in different models

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. IITR Communication No. 2656     

A model for studying pollination and pod development in Brassica napus: the culture of isolated flowers

Summary A technique for cultivating isolated flowers of Brassica napus has been developed. Flowers were harvested at anthesis, the surface of their peduncles was then sterilized and they were cultivated in a hormonefree medium. We used an MS medium supplemented with 3% sucrose as a source of organic carbon. From our experiments, it was concluded that no exogenous growth regulator is required to ensure normal growth and development in vitro. The flowers, and thereafter the pods, can be kept in culture until seed maturity. After 30 days, seed development resulted in three types of seeds: (1) normal, (2) milky and (3) aborted. The results show that the number of seeds per pod was not dependent on the order of flowers on the raceme (except the first 10 flowers and flowers above row 50). Our study supports the validity of this model as an easy tool for studying pollination and early seed development.     

Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term: A valuable model for in vitro toxicity and drug-drug interaction studies

Cultures of primary hepatocytes from various species, including human, are used in several applications during pre-clinical drug development. Their use is however limited by cell survival and conservation of liver-specific functions in vitro. The differentiation status of hepatocytes in culture strongly depends on medium formulation and the extracellular matrix environment. We incubated primary rat hepatocytes for 10 days on collagen monolayer and in collagen sandwich cultures with or without serum. Restoration of polygonal cell shape and formation of functional bile canaliculi-like structures was stable only in serum-free sandwich cultures. Variations in general cell viability, as judged by the cellular ATP content, LDH release or apoptosis, were less pronounced between alternative cultures. The intracellular glutathione content was preserved close to in vivo levels especially in serum-free sandwich cultures. Basal activities of cytochrome P450 enzymes (P450) varied strongly between cultures. There was a minor effect on CYP1A but CYP2B activity was only detectable in the serum-free sandwich culture after 3 days and beyond. CYP2C activity was slightly elevated in both sandwich cultures, whereas CYP3A showed increased levels in both serum-free cultures. Inducibility of these P450s was fully maintained over time in serum-free collagen sandwich only. Gene expression was largely constant over time in serum-free sandwich cultures that was closest to liver. This liver-like property was supported by protein profiling results. Taken together, the serum-free collagen sandwich culture of primary rat hepatocytes maintained liver-like features over 10 days and is therefore a suitable model for long-term toxicity and drug-drug interaction studies.     

A study of the viscoelastic effect in a bone remodeling model

The present paper addresses the following question can a simple regulatory bone remodeling model predict effects of viscosity on the trabecular morphology? For that, we propose an extension of a previous bone remodeling model by taking into account the viscosity properties of the tissue. Zener’s law is used to describe the mechanical behavior of the bone and a specific law of the apparent bone density rate is proposed. Based on stability analysis, numerical simulations are then performed to investigate the viscosity role on simulations of the bone remodeling process. We show that the viscous contribution affects the evolution of the apparent bone density, by slowing down the adaptation process, which seems to be confirmed by simulations with real data obtained from rat tibia.     

Effects of streptozotocin in the male guinea pig: a potential animal model for studying diabetes

The effects of acutely administered streptozotocin in the male guinea pig were studied for a period of 18 days following treatment. A single intracardiac injection of streptozotocin (150 mg/kg) was administered on Day 0. On Day 2, plasma glucose concentrations were not significantly different from control levels. On Day 7 and 18, an oral glucose tolerance test was performed with streptozotocin-treated animals receiving an acute injection of either insulin (18 U/kg, i.m.) or saline 90 minutes prior to glucose loading. On Day 7, streptozotocin-treated animals receiving saline had significantly elevated plasma and urine glucose concentrations at 3 hours after glucose loading when compared to controls. Streptozotocin-treated animals receiving insulin however, had significantly lower plasma glucose concentrations at 3 hours while urinary glucose was equal to control values. The second glucose tolerance test performed on Day 18 yielded similar results. Necropsies were performed on animals that died after Day 6. Lesions found in the streptozotocin-treated animals included: small and irregular pancreatic islets, pyknotic nuclei and degranulation of beta cells, renal proximal tubule swelling and vacuolization, adrenal cortical hyperplasia, hepatocyte vacuolization, and visceral fat atrophy. Animals surviving until Day 18 were sacrificed and found to have significantly elevated kidney and adrenal weights compared to controls. These changes illustrate the effectiveness of streptozotocin in the acute chemical induction of diabetes in an animal model (guinea pig) which, like humans, requires a dietary source of ascorbic acid.     

Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry: a tool for functional genomics

For the rapid identification of functional activity of unknown genes from a sequence database, a new method based on in vitro protein synthesis combined with mass spectrometry was developed. To discriminate their subtle enzymatic activity, in vitro synthesized and one-step purified lipolytic enzymes, such as lip A and lip B from Bacillus subtilis and an unknown protein ybfF from Escherichia coli, were reacted with a mixture of triglycerides with different carbon chain lengths. Using direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of reaction product, all three enzymes were revealed to have strong esterase activity rather than true lipase activity, which has no reactivity on long-chain fatty acids such as triolein. These results were also confirmed by classical color assay using p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as representative lipolytic substrates.     

Structural and Chemical Characterization of a Natural Fracture Surface from 2.8 Kilometers Below Land Surface: Biofilms in the Deep Subsurface

A freshly intersected water-bearing fracture zone from the Mponeng Au mine located in the Witwatersrand Basin, Republic of South Africa was sampled, providing an opportunity to examine the natural, deep subsurface biosphere. The fracture, intersected by an advancing tunnel 2.8 kilometers below land surface, possessed a millimeter thick layer of chlorite group minerals, i.e., chamosite, at the water-mineral interface. Water flowing out from the fracture zone had a temperature of 52°C, pH of 9.16 and Eh of ?263 mV. Using scanning electron microscopy, the water-mineral interface was generally found to be clean, i.e., it did not possess any secondary mineral or dominant organic coatings. Irregular patches (10's of μm ² ) of organic material, however, resembling bacterial exopolysaccharides, occurred in the presence or absence of bacteria. The surface was colonized by highly dispersed individual bacteria or by microcolonies containing up to 5 cells, with an overall cell density of 5 × 10⁴ bacteria cm ^?2 . This biofilm population, although low, was 2 orders of magnitude greater than the bacteria present within the aqueous phase and provides the first direct observation of the sessile population from the terrestrial deep subsurface. Time of Flight-Secondary Ion Mass spectrometry revealed that the fracture surface was actually coated with a thin, i.e., molecular, organic conditioning film over much of its surface that was separate from the exopolysaccharide layers associated with the mineral water interface and with some of the attached cells.     

Brief communication: Sex determination accuracy of the minimum supero-inferior femoral neck diameter in a contemporary rural Guatemalan population

One hundred and fourteen femora (75 male and 39 female) derived from a contemporary rural Guatemalan population were studied to test the ability of the minimum supero-inferior femoral neck diameter as a sex indicator. With the discriminant functions previously developed from North American modern populations, a maximum of only 36% correctly sexed femora was obtained, with correct percentages as low as 4%. A new discriminant function for the Guatemalan rural population is presented, with a total of 89.5% correctly classified individuals. It is suggested that poor physical growth of the rural Guatemalan population, due to a stressful environment, can explain part of the metric differences observed between the North American and rural Guatemalan populations.     

Study of the physicochemical and biological stability of pediocin PA‐1 in the upper gastrointestinal tract conditions using a dynamic in vitro model

Aims: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce pediocin PA‐1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified pediocin PA‐1 under GIT conditions. Methods and Results: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM‐1, comprising four compartments connected by computer‐controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2·7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4‐log reduction after 180 min of digestion, while high viable counts (up to 5 × 10⁷ CFU ml^?1 fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce pediocin at the same level as unstressed cells. The activity of the purified pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. Conclusions: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of pediocin was significantly poor under GIT conditions. Significance and Impact of the Study: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce pediocin PA‐1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified pediocin PA‐1 losses activity during GIT transit; microcapsules could be used to deliver it to the target site.     

A novel, broad-spectrum antitumor compound containing the 1-hydroxycyclohexa-2,5-dien-4-one group: the disclosure of a new antitumor pharmacophore in protoapigenone 1

The synthesis of a new compound 9 containing the 4-hydroxy-2,5-cyclohexadien-1-one system, a key elements toward elucidation of the protoapigenone 1 antitumor pharmacophore, was described. The compound showed potent in vitro antitumor potency with low micromolar IC₅₀’s against breast, ovarian, prostate, liver, pancreas, and blood cancer cell lines tested and could inhibit tumor growth in vivo but no significant impairment of hematopoiesis or immune function was observed. The minimum structural pharmacophore of 1 has now been refined.     

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.     

Effects of wall shear stress and fluid recirculation on the localization of circulating monocytes in a three-dimensional flow model

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall.

Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients.

The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments.

Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.     

Effects of changes in turbidity and phosphate influx on the ecosystem of the Ems estuary as obtained by a computer simulation model

In the Ems estuary the gradients in the concentration of nutrients and in turbidity (the factors that mainly determine the amount of carbon assimilated by phytoplankton) are steep. The effects of changing the turbidity in the estuary and the amount of phosphate discharged by the rivers Ems and Westerwoldsche Aa were analysed, using the simulation model developed by BOEDE (Biological Research Ems-Dollard estuary). The results of several sensitivity runs were compared with the standard run.A 50% reduction of turbidity led to a strong increase in phytoplanktonbiomass, especially in the inner parts of the estuary where turbidity is high. On average, the effects are two to three times larger for the inner part than for the outer part of the estuary. When the turbidity doubles the opposite occurs resulting in a significant decrease of phytoplankton biomass in the upper reaches. In the lower reaches of the estuary a 50% reduction in the river discharge of phosphate is largely compensated for by changes in phosphate transport from the North Sea. This results in a nearly unchanged primary production in the lower reaches as compared with the standard run.In the upper reaches a 50% reduction of phosphate loads results in a strongly reduced primary production.In general, the zooplankton groups (copepods and microzooplankton) are influenced less than the phytoplankton. Benthic fauna is hardly influenced, except for filter feeders; which are strongly affected by the total density of the particles, a parameter which also is directly related to turbidity.     

The stabilizing role of the Sabbath in pre-monarchic Israel:a mathematical model

The three monotheistic cultures have many common institutions and some of them germinated in pre-monarchic Israel. Reasonably, the essential institutions were in place at that starting point; this work explores the possibility that the Sabbath is one of these institutions. Our mathematical examination points to the potential cultural, civic, and social role of the weekly Sabbath, that is, the Sabbath institution, in controlling deviation from social norms. It begins with an analogy between spread of transgression (defined as lack of conformity with social norms) and of biological infection. Borrowing well-known mathematical methods, we derive solution sets of social equilibrium and study their social stability. The work shows how a weekly Sabbath could in theory enhance social resilience in comparison with a similar assembly with a more natural and longer period, say between New Moon and Full Moon. The examination reveals that an efficient Sabbath institution has the potential to ensure a stable organization and suppress occasional appearances of transgression from cultural norms and boundaries. The work suggests the existence of a sharp threshold governed by the “Basic Sabbath Number ש₀”—a critical observance of the Sabbath, or large enough ש₀, is required to ensure suppression of transgression. Subsequently, the model is used to explore an interesting question: how old is the Sabbath? The work is interdisciplinary, combining anthropological concepts with mathematical analysis and with archaeological parallels in regards to the findings.

Electronic supplementary material

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