Chronic Inflammation Alters Production and Release of Glutathione and Related Thiols in Human U373 Astroglial Cells

Neurons rely on glutathione (GSH) and its degradation product cysteinylglycine released by astrocytes to maintain their antioxidant defences. This is particularly important under conditions of inflammation and oxidative stress, as observed in many neurodegenerative diseases including Alzheimer’s disease (AD). The effects of inflammatory activation on intracellular GSH content and the extracellular thiol profile (including cysteinylglycine and homocysteine) of astrocytes were investigated. U373 astroglial cells exposed to IL-1β and TNF-α for up to 96 h showed a dose-dependent increase in IL-6 release, indicative of increasing pro-inflammatory cellular activation. With increasing concentrations of IL-1β and TNF-α (0.01–1 ng/ml), an increase in both intracellular and extracellular GSH levels was observed, followed by a return to control levels in response to higher concentrations of IL-1β and TNF-α. Extracellular levels of cysteinylglycine decreased in response to all concentrations of IL-1β and TNF-α. In contrast, levels of the neurotoxic thiol homocysteine increased in a dose-dependent manner to IL-1β and TNF-α-induced activation. Our results suggest that chronically activated astrocytes in the brain might fail to adequately maintain GSH substrate delivery to neurons, thus promoting neuronal vulnerability. They might also explain the elevated levels of homocysteine found in the brains and serum of patients with AD.     

Fragmentation of decorin, biglycan, lumican and keratocan is elevated in degenerate human meniscus, knee and hip articular cartilages compared with age-matched macroscopically normal and control tissues

Introduction

The small leucine-rich proteoglycans (SLRPs) modulate tissue organization, cellular proliferation, matrix adhesion, growth factor and cytokine responses, and sterically protect the surface of collagen type I and II fibrils from proteolysis. Catabolism of SLRPs has important consequences for the integrity of articular cartilage and meniscus by interfering with their tissue homeostatic functions.

Methods

SLRPs were dissociatively extracted from articular cartilage from total knee and hip replacements, menisci from total knee replacements, macroscopically normal and fibrillated knee articular cartilage from mature age-matched donors, and normal young articular cartilage. The tissue extracts were digested with chondroitinase ABC and keratanase-I before identification of SLRP core protein species by Western blotting using antibodies to the carboxyl-termini of the SLRPs.

Results

Multiple core-protein species were detected for all of the SLRPs (except fibromodulin) in the degenerate osteoarthritic articular cartilage and menisci. Fibromodulin had markedly less fragments detected with the carboxyl-terminal antibody compared with other SLRPs. There were fewer SLRP catabolites in osteoarthritic hip than in knee articular cartilage. Fragmentation of all SLRPs in normal age-matched, nonfibrillated knee articular cartilage was less than in fibrillated articular cartilage from the same knee joint or total knee replacement articular cartilage specimens of similar age. There was little fragmentation of SLRPs in normal control knee articular cartilage. Only decorin exhibited a consistent increase in fragmentation in menisci in association with osteoarthritis. There were no fragments of decorin, biglycan, lumican, or keratocan that were unique to any tissue. A single fibromodulin fragment was detected in osteoarthritic articular cartilage but not meniscus. All SLRPs showed a modest age-related increase in fragmentation in knee articular and meniscal cartilage but not in other tissues.

Conclusion

Enhanced fragmentation of SLRPs is evident in degenerate articular cartilage and meniscus. Specific decorin and fibromodulin core protein fragments in degenerate meniscus and/or human articular cartilage may be of value as biomarkers of disease. Once the enzymes responsible for their generation have been identified, further research may identify them as therapeutic targets.     

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.     

Male and female contributions to behavioral isolation in darters as a function of genetic distance and color distance

Determining which reproductive isolating barriers arise first between geographically isolated lineages is critical to understanding allopatric speciation. We examined behavioral isolation among four recently diverged allopatric species in the orangethroat darter clade (Etheostoma: Ceasia). We also examined behavioral isolation between each Ceasia species and the sympatric rainbow darter Etheostoma caeruleum. We asked (1) is behavioral isolation present between allopatric Ceasia species, and how does this compare to behavioral isolation with E. caeruleum, (2) does male color distance and/or genetic distance predict behavioral isolation between species, and (3) what are the relative contributions of female choice, male choice, and male competition to behavioral isolation? We found that behavioral isolation, genetic differentiation, and male color pattern differentiation were present between allopatric Ceasia species. Males, but not females, discerned between conspecific and heterospecific mates. Males also directed more aggression toward conspecific rival males. The high levels of behavioral isolation among Ceasia species showed no obvious pattern with genetic distance or male color distance. However, when the E. caeruleum was included in the analysis, an association between male aggression and male color distance was apparent. We discuss the possibility that reinforcement between Ceasia and E. caeruleum is driving behavioral isolation among allopatric Ceasia species.     

Cue-Recruitment for Extrinsic Signals after Training with Low Information Stimuli

Cue-recruitment occurs when a previously ineffective signal comes to affect the perceptual appearance of a target object, in a manner similar to the trusted cues with which the signal was put into correlation during training [1], [2]. Jain, Fuller and Backus [3] reported that extrinsic signals, those not carried by the target object itself, were not recruited even after extensive training. However, recent studies have shown that training using weakened trusted cues can facilitate recruitment of intrinsic signals [4]–[7]. The current study was designed to examine whether extrinsic signals can be recruited by putting them in correlation with weakened trusted cues. Specifically, we tested whether an extrinsic visual signal, the rotary motion direction of an annulus of random dots, and an extrinsic auditory signal, direction of an auditory pitch glide, can be recruited as cues for the rotation direction of a Necker cube. We found learning, albeit weak, for visual but not for auditory signals. These results extend the generality of the cue-recruitment phenomenon to an extrinsic signal and provide further evidence that the visual system learns to use new signals most quickly when other, long-trusted cues are unavailable or unreliable.     

NmeCas9 is an intrinsically high-fidelity genome-editing platform

Background

The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.

Results

Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N₄GATT-3′), for NmeCas9 genome editing in human cells.

Conclusions

Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

     

Isotopic evidence for breastfeeding and possible adult dietary differences from Late/Sub-Roman Britain

Historical documents indicate that breastfeeding and weaning practices have fluctuated in England through history. In order to obtain evidence for general breastfeeding patterns in Late/Sub-Roman Britain, stable carbon and nitrogen isotope values were measured in juvenile and adult skeletons (n = 87) from the cemetery of Queenford Farm, Dorchester-on-Thames, Oxfordshire. As the site contained few individuals between 0-1.5 years of age, it was not possible to determine the initial timing for the introduction of weaning foods. Between ages 2-4 years, the mean +/- SD delta(13)C results (-20.2 +/- 0.3 per thousand) are significantly more negative (t = -4.03, P < 0.001) compared to adult females (-19.7 +/- 0.3 per thousand). This is interpreted as evidence of a different diet being fed to children during weaning. After age 2, the delta(15)N values gradually decline, indicating complete cessation of breastfeeding by 3-4 years. Among adults, stature (males = 1.68 +/- 0.06 m; females = 1.58 +/- 0.07 m) and sexual dimorphism (106) were low, suggesting that the population was possibly under environmental stress. The delta(13)C results for adults are similar, but females show a small but statistically significantly (t = -2.86, P < 0.01) lower mean delta(15)N value (9.9 +/- 0.9 per thousand) compared to males (10.6 +/- 0.5 per thousand). These lower female delta(15)N values possibly reflect the different physiology of the sexes (pregnancy and/or lactation) or the reduced consumption of animal/fish protein by women, and this may have been influenced by individual preference, family needs, or societal values of the era.     

Analysis of DNA Sequencing Reaction Products Made with 7-Halo-7-deaza-2′-deoxyguanosine-5′-triphosphate

Abstract

7-Chloro- and 7- Iodo-7-Deaza-2′-Deoxyguanosine-5′-Triphosphates were employed as substrates replacing dGTP, dITP, or 7-Deaza-2′-Deoxyguanosine-5′-Triphosphate in sequencing reactions with Thermo Sequenase^TM. Analysis of the reaction products by denaturing gel electrophoresis indicates DNA containing these halogenated analogs can form strong secondary structures.