Computational evaluation of oxygen and shear stress distributions in 3D perfusion culture systems: macro-scale and micro-structured models

We present a combined macro-scale/micro-scale computational approach to quantify oxygen transport and flow-mediated shear stress to human chondrocytes cultured in three-dimensional scaffolds in a perfusion bioreactor system. A macro-scale model was developed to assess the influence of the bioreactor design and to identify the proper boundary conditions for the micro-scale model. The micro-scale model based on a micro-computed tomography (microCT) reconstruction of a poly(ethylene glycol terephthalate)/poly(butylene terephthalate) (PEGT/PBT) foam scaffold, was developed to assess the influence of the scaffold micro-architecture on local shear stress and oxygen levels within the scaffold pores. Experiments were performed to derive specific oxygen consumption rates for constructs perfused under flow rates of 0.3 and 0.03 ml min(-1). While macro-scale and micro-scale models predicted similar average oxygen levels at different depths within the scaffold, microCT models revealed small local oxygen variations within the scaffold micro-architecture. The combined macro-scale/micro-scale approach indicated that 0.3 ml min(-1), which subjected 95% of the cells to less than 6.3 mPa shear, would maintain the oxygen supply throughout the scaffold above anoxic levels (>1%), with 99.5% of the scaffold supplied with 8-2% O(2). Alternatively, at 0.03 ml min(-1), the macro-scale model predicted 6% of the cells would be supplied with 0.5-1% O(2), although this region of cells was confined to the periphery of the scaffold. Together with local variations predicted by the micro-scale model, the simulations underline that in the current model system, reducing the flow below 0.03 ml min(-1) would likely have dire consequences on cell viability to pronounced regions within the engineered construct. The presented approach provides a sensitive tool to aid efficient bioreactor optimization and scaffold design.     

19F]-1H heteronuclear nuclear Overhauser effect studies of the acyl chain-binding site of acyl carrier protein

[19F]-1H heteronuclear difference nuclear Overhauser effect experiments are performed on a sample of 5,5-difluorohexanoyl acyl carrier protein from Escherichia coli. Interaction of the fluorines at the 5-position of the acyl chain with protons on methyl groups of isoleucine 54 and alanine 59 is clearly indicated. The covalent attachment of the acyl chain via a prosthetic group to serine 36 and the known alpha-helix which exists from residues 36 to 51 greatly restrict the structural models which would allow acyl chain contact with residues 54 and 59.     

Testosterone: the culprit for producing splenocyte immune depression after trauma hemorrhage

Studies indicate that, whereas immune functions in males aredepressed, they are enhanced in females after trauma hemorrhage. Moreover, castration of male mice (i.e., androgen depletion) before trauma hemorrhage prevented the depression of cell-mediated immunity. Nonetheless, it remains unknown whether or not testosterone per se isresponsible for producing the immune depression. To study this, femaleC3H/HeN mice (n = 7 animals/group)were pretreated with 5-dihydrotestosterone (DHT) or vehicle for 19 days, then subjected to laparotomy (e.g., trauma) and hemorrhagic shock(blood pressure 35 ± 5 mmHg for 90 min) followed by fluidresuscitation or sham operation. Nontreated males underwent eithertrauma hemorrhage or sham operation. Twenty-four hours thereafter,splenocyte immune functions as well as plasma DHT, estradiol, andcorticosterone levels were measured. DHT-pretreated females hadsignificantly (P < 0.05) increasedDHT levels, comparable to those seen in males. Conversely, estradiollevels in such females were similar to control males. Splenocyteproliferation as well as interleukin-2 and interleukin-3 release werenot depressed in vehicle-treated females, whereas it was in DHT-treatedfemales after trauma hemorrhage, comparable to hemorrhaged males. Thushigh testosterone and/or low estradiol levels appear to beresponsible for producing splenocyte immune depression in males aftertrauma hemorrhage. Agents that block testosterone receptors or increaseestradiol levels may therefore be helpful in improving depressed immunefunctions in male trauma patients.

     

Secondary metabolites from the aerial parts of Salvia palaestina Bentham

Three sesterterpenes (1-3), one triterpene (4) and five diterpenes (5-9) were isolated from the aerial parts of Salvia palaestina Bentham (Lamiaceae), together with two sesquiterpenes, 10 known diterpenes, three triterpenes, and rosmarinic acid. Their structural elucidation was accomplished by extensive spectroscopic methods including 1D ((1)H, (13)C, (13)C DEPT, TOCSY, NOESY) and 2D NMR experiments (DQF-COSY, HSQC, HMBC, ROESY) as well as ESIMS analysis and chemical analysis.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Adiposity Predicts Cognitive Decline in Older Persons with Diabetes: A 2-Year Follow-Up

Background

The mechanisms related to cognitive impairment in older persons with Type 2 diabetes (DM) remains unclear. We tested if adiposity parameters and body fat distribution could predict cognitive decline in older persons with DM vs. normal glucose tolerance (NGT).

Methodology

693 older persons with no dementia were enrolled: 253 with DM in good metabolic control; 440 with NGT (age range:65–85 years). Longitudinal study comparing DM and NGT individuals according to the association of baseline adiposity parameters (body mass index (BMI), waist-hip-ratio (WHR), waist circumference (WC) and total body fat mass) to cognitive change (Mini Mental State Examination (MMSE), a composite score of executive and attention functioning (CCS) over time.

Findings

At baseline, in DM participants, MMSE correlated with WHR (β = −0.240; p = 0.043), WC (β = −0.264; p = 0.041) while CCS correlated with WHR (β = −0.238; p = 0.041), WC (β = −0.326; p = 0.013) after adjusting for confounders. In NGT subjects, no significant correlations were found among any adiposity parameters and MMSE, while CCS was associated with WHR (β = −0.194; p = 0.036) and WC (β = −0.210; p = 0.024). Participants with DM in the 3^rd tertile of total fat mass showed the greatest decline in cognitive performance compared to those in 1^st tertile (tests for trend: MMSE(p = 0.007), CCS(p = 0.003)). Logistic regression models showed that 3^rd vs. 1^st tertile of total fat mass, WHR, and WC predicted an almost two-fold decline in cognitive function in DM subjects at 2^nd yr (OR 1.68, 95%IC 1.08–3.52).

Conclusions

Total fat mass and central adiposity predict an increased risk for cognitive decline in older person with DM.     

Chromosomal evolution in naked catfishes (Bagridae,Siluriformes): A comparative chromosome mapping study

We analyzed the distribution of repetitive DNA sequences on the chromosomes of nine species of the Bagridae from Thailand, i.e., Hemibagrus filamentus; H. nemurus; H. wyckii; H. wyckioides; Mystus atrifasciatus; M. multiradiatus; M. mysticetus; M. bocourti and Pseudomystus siamensis. Two classes of microsatellites and one transposable element (TE) were mapped by fluorescence in situ hybridization. The distribution of the repetitive sequences was comparatively analyzed in view to investigate their contribution in the chromosomal evolution of this fish group. In all species the microsatellites (CA)₁₅ and (GA)₁₅ are abundantly distributed in all chromosomes, usually in the telomeric regions. The retrotransposable element Rex 1 is widely distributed over the whole genome including heterochromatin and euchromatin, but with an unexpected accumulation in one chromosome pair in some species. In fact, some species–specific patterns could be observed considering both microsatellites and TE distribution. The results demonstrated that the compartmentalization of repeated elements is not simply restricted to heterochromatic regions, as it has been postulated in the first concepts of the genomic organization of repetitive elements in genomes. Moreover, the organization of these repeats seems to reflect their intense and specific evolutionary pathway, providing new insights about the chromosomal evolution in the Bagridae.