In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci

 

Shed cells or disrupted parts of the biofilm may enter the circulation causing serious and very hard to treat biofilm-associated infections. The activity of antimicrobial agents against the shed cells/disrupted biofilms is largely unknown.

Methods

We studied the in vitro susceptibility of intact and disrupted biofilms of thirty clinical isolates of methicillin-resistant and methicillin–susceptible Staphylococcus aureus (MRSA and MSSA) and Staphylococcus epidermidis to vancomycin, quinupristin/dalfopristin, and linezolid and compared it to that of the suspended (planktonic) cells.

Results

Bacteria in the disrupted biofilms were as resistant as those in the intact biofilms at the minimum inhibitory concentrations of the antibiotics. At higher concentrations, bacteria in the disrupted biofilms were significantly (P < 0.001) less resistant than those in the intact biofilms but more resistant than the planktonic cells. Quinupristin/dalfopristin showed the best activity against cells of the disrupted biofilms at concentrations above MICs and vancomycin, at 500 and 1,000 μg/ml, was significantly more active against the biofilms of MRSA and S. epidermidis

Conclusion

The difficulty of treating biofilm-associated infections may be attributed not only to the difficulty of eradicating the biofilm focus but also to the lack of susceptibility of cells disrupted from the biofilm to antimicrobial agents.     

Differentiation promoting factors induced in Acanthamoeba by inhibitors of mitochondrial macromolecule synthesis

Acanthamoeba castellanii (line OS4) differentiates into a cyst when treated with mitochondrial inhibitors berenil, ethidium bromide, erythromycin, and chloramphenicol. The process is a function of cell density at the time of inhibition: Early log phase cells yield 0–20% cysts, whereas mid-log phase cells yield up to 55, 65, or 95% cysts when treated with chloramphenicol or erythromycin, ethidium bromide, or berenil, respectively. Medium from encysting mid-log phase cells contains an encystment enhancing activity (EEA) which, when transferred to low-density early log phase cultures, causes high levels of encystment. EEA has a nominal molecular weight of between 1000 and 10,000. It is resistant to nucleases, proteases, amylase, boiling, and 1 N NaOH, 25°C, but is inactivated by snake venom phosphodiesterase, autoclaving, and acidic or harsher alkaline treatments. Other lines, OS1 and OS5, which do not encyst well at any cell density in response to the four inhibitors or to EEA, nevertheless produce EEAs to which OS4 can respond. OS4 has been adapted to growth in a chemically defined medium; when starved for glucose and acetate, the cells produce a phosphodiesterase-sensitive EEA. A similar activity can be obtained from mitochondria treated with berenil in vitro. Several lines of evidence suggest that EEAs from the various sources are the same. We propose that they may be of mitochondrial origin and may act as developmental effectors under conditions unfavorable for cell growth.     

植被重建对露天煤矿排土场土壤酶活性的影响

植被重建是露天煤矿排土场生态恢复的关键措施,深入了解植被建设对土壤酶活性的影响,对于合理选择适宜于矿区生态恢复的人工植被和加速矿区土壤生态恢复具有重要意义。通过野外调查采样和室内分析,研究了黑岱沟露天煤矿排土场植被重建和恢复对浅层(0—20 cm)土壤酶活性(包括3种氧化还原酶:过氧化氢酶、多酚氧化酶、脱氢酶,4种水解酶:蔗糖酶、脲酶、磷酸酶、纤维素酶)的影响。结果表明:相比未进行植被建设的新排土场裸地,植被重建显著改善了土壤酶活性和理化性质,建植18a后土壤酶活性可恢复到天然植被区的65%—76%,水解酶恢复速率(平均为86.9%)快于氧化还原酶(平均为42.7%),其中土壤磷酸酶恢复速率最快(平均为天然植被区的154.7%),其次为蔗糖酶(74.3%)、纤维素酶(59.9%)、脲酶(58.5%)、过氧化氢酶(52.1%)和脱氢酶(38.1%),多酚氧化酶恢复最慢(为37.8%)。植被恢复进程中,建植10a期土壤酶活性年均恢复速率最快(平均为6.0%/a),15a变缓(4.8%/a),18a迅速降低(3.2%/a)。同时植被配置类型对土壤酶活性影响显著,土壤酶活性与土壤主要理化因子具有较高的相关性。上述结果反映了植被重建能显著改善矿区排土场的土壤酶活性,植被恢复进程中水解酶恢复速率快于氧化还原酶,恢复初期快于后期,但土壤酶活性的恢复需要一个漫长的过程。     

Establishing a laboratory model of dental unit waterlines bacterial biofilms using a CDC biofilm reactor

In this study, a laboratory model to reproduce dental unit waterline (DUWL) biofilms was developed using a CDC biofilm reactor (CBR). Bacteria obtained from DUWLs were filtered and cultured in Reasoner’s 2A (R2A) for 10 days, and were subsequently stored at ?70°C. This stock was cultivated on R2A in batch mode. After culturing for five days, the bacteria were inoculated into the CBR. Biofilms were grown on polyurethane tubing for four days. Biofilm accumulation and thickness was 1.3 × 10⁵ CFU cm^?2 and 10–14 μm respectively, after four days. Bacteria in the biofilms included cocci and rods of short and medium lengths. In addition, 38 bacterial genera were detected in biofilms. In this study, the suitability and reproducibility of the CBR model for DUWL biofilm formation were demonstrated. The model provides a foundation for the development of bacterial control methods for DUWLs.     

Development of A Loop-Mediated Isothermal Amplification (LAMP) for the Detection of F5 Fimbriae Gene in Enterotoxigenic Escherichia coli (ETEC)

The objective of this study was to establish a loop-mediated isothermal amplification (LAMP) method for the detection of F5 fimbriae gene in Enterotoxigenic Escherichia coli. A set of four primers were designed based on the conservative sequence of coding F5 fimbriae. Temperature and time condition, specificity test, and sensitivity test were performed with the DNA of Escherichia coli (F5+). The results showed that the optimal reaction condition for LAMP was achieved at 61 °C for 45 min in a water bath. Ladder-like products were produced with those F5-positive samples by LAMP, while no product was generated with other negative samples. The assay of LAMP had a detection limit equivalent to 72 cfu/tube, which was more sensitive than PCR (7.2 × 10² cfu/tube). The agreement rate between LAMP and PCR was 100 % in detecting simulation samples. Thus, the LAMP assay may be a new method for rapid detection of F5 fimbriae gene of ETEC.     

Local effects of forest fragmentation on diversity of aquatic insects in tropical forest streams: implications for biological conservation

The influence of forest fragmentation (habitat isolation) on biological and ecological diversity of aquatic insects was investigated in streams of fragmented forests in Hulu Gombak (6 streams) and Gunung Angsi (5 streams) and un-fragmented forest of Berembun (6 streams) in peninsular Malaysia. Several environmental parameters including canopy cover, DO, temperature and pH differed significantly among the three catchments (P < 0.05). We found that taxonomic richness in Berembun forest was significantly different from Gunung Angsi (P < 0.05), but not with Hulu Gombak forests (P > 0.05). Nestedness pattern that measures the effect of habitat isolation on taxonomic assemblages showed that aquatic insect’s community in un-fragmented forest (Berembun) was less nested (T = 54.4), indicating high diversity compared to highly nested (less diverse) in the two fragmented forests (Hulu Gombak, T = 30.45 and Gunung Angsi, T = 35.45). Taxa similarity in Berembun streams was negatively correlated with the geographical distance among streams (Mantel test, r = ? 0.462, P < 0.05). Such correlation was absent in both Gunung Angsi and Hulu Gombak streams. Forest fragmentation in Hulu Gombak and Gunung Angsi measured as the distance of the forests from the nearest forested area had negative effect on aquatic insects diversity (r ² = ? 0.149, P < 0.05), but not on their abundances (r ² = 0.003, P > 0.05). We concluded that local habitat conditions were the most important in shaping the aquatic insects community among streams of both unfragmented and fragmented forests.     

Evidence for consumer regulation of biofilm–nutrient interactions among hardwater streams (Pennsylvania, USA)

Experimental studies evaluating the simultaneous effects of consumers, nutrients, and other biotic/abiotic factors on intact, natural food webs are rare, particularly among ecosystems of varying trophic conditions. We conducted a series of in situ studies that used nutrient-diffusing substrata with nitrogen (N) and phosphorus (P) concentrations in a full factorial design in three temperate, limestone streams in Pennsylvania across a trophic gradient (mesotrophic, eutrophic, and hypereutrophic streams). We assessed differences in algal and macroinvertebrate biomass, taxonomic composition, and functional groups relative to amended nutrients across the trophic gradient; as such, these results facilitated predictions about regulators of food web structure. All factors varied significantly among the streams (e.g., algal biomass P = 0.005, macroinvertebrate biomass P < 0.001, algal diversity P = 0.006, macroinvertebrate diversity P < 0.001, algal group P < 0.001, macroinvertebrate guilds P < 0.001); the streams, however, did not exhibit simple responses to nutrient amendment. Algal and macroinvertebrate biomass and diversity responded greatest in the mesotrophic stream while grazing seemed to be a strong factor preventing algal nutrient response in the eutrophic and hypereutrophic streams. Brillouin’s Evenness Index was most influenced by nutrient amendment (nutrient effect on algae and macroinvertebrates P = 0.021). As such, we concluded that biomass and diversity were mediated by complexity within intermediate trophic levels.     

Effects of galactose on direct and indirect pathway estimates of hepatic glycogen synthesis

Hepatic glycogen is formed by direct and indirect pathways whose activities reflect altered nutrition or disease. Direct/indirect pathway measurements often involve test meals where ～10% of carbohydrate is galactose, but its effects on direct/indirect pathway estimates are unknown. Therefore, direct/indirect pathway contributions in 24-h fasted rats given 2 g/kg 100% glucose (GLU, n=6) or 90% glucose–10% galactose (GLU+GAL, n=6) were measured by [U-¹³C]glucose dilution and by position-5/position-2 glycogen enrichment (H5/H2) from 2H₂O. For GLU+GAL, galactose glycogenesis was independently measured with [1-¹³C]galactose. Glycogenesis was equivalent in both groups but for GLU+GAL, 23±4% of glycogen was derived from galactose. [U-¹³C]glucose reported a 30±3% direct pathway contribution to glycogenesis for GLU but only 20±3% for GLU+GAL (p=0.012 vs. GLU). H5/H2 yielded identical direct pathway estimates (32±3% GLU, 29±6% GLU+GAL). Thus, galactose glycogenesis was undetected by H5/H2 while [U-¹³C]glucose reported a reduced direct/indirect pathway ratio. With [1-¹³C]galactose also present, correct glycogenic source contributions were obtained.     

Soil oxidases recovered faster than hydrolases in a 50-year chronosequence of desert revegetation

Aims

Desert characterized by alkaline soil with low organic matter and nutrients has a high soil oxidative potential. We hypothesized that oxidase activities would recover faster than hydrolases during the succession of sand-fixing community.

Methods

Sand dunes stabilized in different years, including a moving sand dune and a steppe at the southeastern fringe of the Tengger Desert, were selected to investigate restoration of extracellular enzyme activities (EEAs) in a 50-year chronosequence.

Results

Oxidases showed significantly higher activities than hydrolases at all ten studied sites and EEAs exibited a decreasing trend from catalase, phenol oxidase, sucrase, urease, alkaline phosphatase, α-Amylase to cellulase. After 50 years of revegetation, most EEAs in topsoil recovered to 50–83% of that of the steppe except for urease. Oxidase activities recovered earlier and faster than hydrolases, while hydrolases activities attained the fastest recovery at 19–25 years in the 50-year chronosequence.

Conclusions

Recovery of EEAs was modulated by the succession of the sand-fixed community: oxidases activities exhibited peak recovery rates at the stage when shrubs dominated the community, while recovery of hydrolases activities appeared to be mainly regulated by biological soil crusts and annual plants.     

Bacterial Versus Archaeal Origin of Extracellular Enzymatic Activity in the Northeast Atlantic Deep Waters

We determined the total and dissolved extracellular enzymatic activity (EEA) of α-glucosidase and β-glucosidase (AGase and BGase), alkaline phosphatase (APase) and leucine aminopeptidase (LAPase) activities in the epi-, meso- and bathypelagic waters of the subtropical Northeast Atlantic. EEA was also determined in treatments in which bacterial EEA was inhibited by erythromycin. Additionally, EEA decay experiments were performed with surface and deep waters to determine EEA lifetimes in both water masses. The proportion of dissolved to total EEA (66–89 %, 44–88 %, 57–82 % and 86–100 % for AGase, BGase, APase and LAPase, respectively) was generally higher than the cell-associated (i.e., particulate) EEA. The percentage of dissolved to total EEA was inversely proportional to the percentage of erythromycin-inhibited to total EEA. Since erythromycin-inhibited plus dissolved EEA equaled total EEA, this tentatively suggests that cell-associated EEA in the open oceanic water column is almost exclusively of bacterial origin. The decay constants of dissolved EEA were in the range of 0.002–0.048 h^?1 depending on the type of extracellular enzyme, temperature and depth in the water column. Although dissolved EEA can have different origins, the major contribution of Bacteria to cell-associated EEA and the long life-time of dissolved EEA suggest that Bacteria—and not mesophilic Archaea—are essentially the main producers of EEA in the open subtropical Northeast Atlantic down to bathypelagic layers.     

Endogenous Content and Release of [3H]-GABA and [3H]-Glutamate in the Spinal Cord of Chronically Undernourished Rat

The aim of this study was to determine the effect of chronic undernutrition on the content and release of γ-amino butyric acid (GABA) and glutamate (GLU) transmitters in the rat spinal cord. The release of [³H]-GABA and [³H]-GLU was determined by radioactive liquid scintillation techniques, and the concentrations of GABA and GLU in spinal cord preparations from control and undernourished young rats (50–60 days old) were measured by reverse-phase HPLC. The GABA and GLU contents in the lumbar spinal dorsal horn (L6 segment) were significantly lower in undernourished rats relative to control rats (22.2 ± 3.7 and 10.7 ± 1.9 %, respectively; P < 0.05). Spinal cord blocks from undernourished animals also showed lower rates of [³H]-GABA and [³H]-GLU release than controls (27.6 ± 3.5 and 12.8 ± 2.5 %, respectively; P < 0.01). We propose that the decreases in GLU content and release are consistent with a reduced activation of either afferent fibers, spinal glutaminergic neurons, or both. Furthermore, we propose that the decreased content and release of GABA in undernourished animals are related to a depression in pre- and post-synaptic inhibition. In addition, we hypothesize that the reductions in GABA content and release serve as compensatory mechanisms to counterbalance decreases in sensory transmission and GLU content in the spinal cord of the chronically undernourished rat.     

Microbial ecoenzyme stoichiometry,nutrient limitation,and organic matter decomposition in wetlands of the conterminous United States

Microbial respiration (R_m) and ecoenzyme activities (EEA) related to microbial carbon, nitrogen, and phosphorus acquisition were measured in 792 freshwater and estuarine wetlands (representing a cumulative area of 217,480 km²) across the continental United States as part of the US EPA’s 2011 National Wetland Condition Assessment. EEA stoichiometry was used to construct models for and assess nutrient limitation, carbon use efficiency (CUE), and organic matter decomposition (? k). The wetlands were classified into ten groups based on aggregated ecoregion and wetland type. The wetlands were also assigned to least, intermediate, and most disturbed classes, based on the extent of human influences. Ecoenzyme activity related to C, N and P acquisition, R_m, CUE, and ? k differed among ecoregion–wetland types and, with the exception of C acquisition and ? k, among disturbance classes. R_m and EEA were positively correlated with soil C, N and P content (r = 0.15–0.64) and stoichiometry (r = 0.15–0.48), and negatively correlated with an index of carbon quality (r = ? 0.22 to ? 0.39). EEA stoichiometry revealed that wetlands were more often P- than N-limited, and that P-limitation increases with increasing disturbance. Our enzyme-based approach for modeling C, N, and P acquisition, and organic matter decomposition, all rooted in stoichiometric theory, provides a mechanism for modeling resource limitations of microbial metabolism and biogeochemical cycling in wetlands. Given the ease of collecting and analyzing soil EEA and their response to wetland disturbance gradients, enzyme stoichiometry models are a cost-effective tool for monitoring ecosystem responses to resource availability and the environmental drivers of microbial metabolism, including those related to global climate changes.     

First pollen record for Ancyloscelis apiformes (Apidae: Emphorini) in Central Amazon

Abstract

The first pollen study for the solitary bee Ancyloscelis apiformes (Emphorini) in a nest aggregation in the Central Amazon was made in October 2012 at the Sucupira apiary in east Manaus, Amazonas, at 03° 00? 05″ S, 59° 51? 05″ W. A total of 15 samples/nests (N1–N15) were analysed, including intact pollen loads (stored inside the nest) and post-emergence residues, which were acetolysed and mounted in glycerine gelatin. From each sample, 500 pollen grains were counted to determine the pollen frequencies and diversity (H′) and evenness (J′) indices. The most representative pollen types were Bonamia ferruginea (N1 = 95.6% and N2 = 96.00%) and the Ipomoea (N3 = 96.40%), both of which belong to the Convolvulaceae. In addition, Byrsonima crispa (N10 = 93.40%) and Malpighia glabra (N7 = 90.20% and N9 = 97.60%), representatives of the Malpighiaceae, were classified as temporary specialisation events with frequencies above 90%. In this study, A. apiformes, which is considered an oligolectic bee species, presented a polylectic tendency since it collected large percentages of pollen types from plants in distinct families that were not phylogenetically related.     

Development of Loop-Mediated Isothermal Amplification (LAMP) for Universal Detection of Enteroviruses

Enteroviruses are found in most environments and cause several diseases in humans. Loop-mediated isothermal amplification (LAMP) was adapted and evaluated for the rapid detection of enteroviruses. Based on the highly conserved 5′ untranslated region (5′-UTR) of the human enteroviruses (HEVs), particularly human enterovirus A (HEV-A) and HEV-B, a set of universal primers was designed. The LAMP amplification was carried out under isothermal conditions at 61 °C, depending on the template concentration results were obtained within 45–90 min. The detection limits were found to be 10¹ copies of cloned enterovirus 71 fragments, more sensitive than conventional PCR. Nine water samples collected from drinking water sources during three seasons and 19 stool specimens collected from HFMD patients were analyzed. By using the LAMP assay, the majority of samples was tested positive, 9/9 (100 %) and 18/19 (94.7 %), respectively. LAMP is a practical method for the rapid detection of enteroviruses in environmental and clinical samples.     

Extracellular enzymatic activities of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina)

As part of a project aimed at the selection of cold-adapted yeasts expressing biotechnologically interesting features, the extracellular enzymatic activity (EEA) of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina) was investigated. Ninety-one basidiomycetous yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Dioszegia, Mrakia, Rhodotorula, Rhodosporidium, Sporobolomyces, Sporidiobolus, Cystofilobasidium, and Udeniomyces) were screened for extracellular amylolytic, proteolytic, lipolytic, esterasic, pectinolytic, chitinolytic, and cellulolytic activities. Over 15% of the strains exhibited three or more different EEAs at 4 degrees C and more than 63% had at least two EEAs at the same temperature. No chitinolytic or cellulolytic activities were detected at 4 and 20 degrees C. Cell-free supernatants exhibited significantly higher (P < 0.01) protease and lipase activities at < or = 10 degrees C, or even at 4 degrees C. In light of these findings, cold environments of Patagonia (Argentina) may be considered a potential source of cold-adapted yeasts producing industrially relevant cold-active enzymes.     

A rapid,simple and sensitive loop-mediated isothermal amplification method to detect Anaplasma bovis in sheep and goats samples

A loop-mediated isothermal amplification (LAMP) technique has been widely used in detecting the nucleic acid of various pathogenic bacteria. In this study, a set of four LAMP primers was designed to specifically test Anaplasma bovis. The LAMP assay was performed at 62 °C for 60 min in a water bath. The specificity was confirmed by amplifying A. bovis isolate, while no cross reaction was observed with other five pathogens (Anaplasma bovis, Anaplasma phagocytophilum, Theileria luwenshuni, Babesia motasi and Schistosoma japonicum). The sensitivity of LAMP was 5 × 10⁰ copies/μL, 100 times more than that of conventional PCR (5 × 10² copies/μL). Of 120 blood DNA extracted from sheep and goats field samples, 81 (67.5%), 22 (18.3%) and 43 (35.8%) were positively detected by LAMP, conventional PCR and nested PCR, respectively. The findings indicated that the developed LAMP assay is a new convenient tool for rapid and cost-effective detection of A. bovis.     

Cryopreserved amniotic membrane as transplant allograft: viability and post-transplant outcome

Amniotic membrane (AM) transplantation is increasingly used in ophthalmological and dermatological surgeries to promote re-epithelialization and wound healing. Biologically active cells in the epithelial and stromal layers deliver growth factors and cytokines with anti-inflammatory, anti-bacterial, anti-immunogenic and anti-fibrotic properties. In this work, confocal microscopy was used to show that our cryopreservation protocol for AM yielded viable cells in both the stromal and epithelial layers with favorable post-transplant outcome. AM was obtained from Caesarean-section placenta, processed into allograft pieces of different sizes (3 cm × 3 cm, 5 cm × 5 cm, and 10 cm × 10 cm) and cryopreserved in 10 % dimethyl sulfoxide using non-linear controlled rate freezing. Post-thaw cell viability in the entire piece of AM and in the stromal and epithelial cell layers was assessed using a dual fluorescent nuclear dye and compared to hypothermically stored AM, while surveys from surgical end-users provided information on post-transplant patient outcomes. There was no significant statistical difference in the cell viability in the entire piece, epithelial and stromal layers regardless of the size of allograft piece (p = 0.092, 0.188 and 0.581, respectively), and in the entire piece and stromal layer of hypothermically stored versus cryopreserved AM (p = 0.054 and 0.646, respectively). Surgical end-user feedback (n = 49) indicated that 16.3 % of AM allografts were excellent and 61.2 % were satisfactory. These results support the expanded clinical use of different sizes of cryopreserved AM allografts and address the issue of orientation of the AM during transplant for the treatment of dermatological defects and ocular surface disorders.     

Maternal Behavior,Posture Change,and Production Performance of Lactating Sows Housed in an Enriched Environment

This study aimed to evaluate whether adding straw to a loose-farrowing house promotes maternal functions and production. Forty-eight sows (Landrace× Large White) were housed in either a farrowing pen without straw (C, n = 24) or with straw (S, n = 24). Behaviors were observed using video recordings and were statistically analyzed. Lateral recumbency was higher and standing was lower in S compared with C (p = .034 and p = .020, respectively), and lateral recumbency to other postures, ventral to lateral recumbency and standing to lying were markedly lower in S than C (p = .014, p = .025 and p = .023, respectively) on Day 1 postpartum. However, except piglet losses during the first three days postpartum (p = .032), piglet weight on Day 21 (p = .037), and piglet weaning weight (p = .020), other production performances were not significantly different between the two groups during the whole experimental period (p ?.05). The results suggest the enrichment of a farrowing pen with straw has important beneficial effects on sow and piglet welfare and improves piglet survival rates.     

The effects of 12-week progressive strength training on strength,functional capacity,metabolic biomarkers,and serum hormone concentrations in healthy older women: morning versus evening training

ABSTRACT

Previous findings suggest that performing strength training (ST) in the evening may provide greater benefit for young individuals. However, this may not be optimal for the older population. The purpose of this study was to compare the effects of a 12-week ST program performed in the morning vs. evening on strength, functional capacity, metabolic biomarker and basal hormone concentrations in older women. Thirty-one healthy older women (66 ± 4 years, 162 ± 4 cm, 75 ± 13 kg) completed the study. Participants trained in the morning (M) (07:30, n = 10), in the evening (E) (18:00, n = 10), or acted as a non-training control group (C) (n = 11). Both intervention groups performed whole-body strength training with 3 sets of 10–12 repetitions with 2–3 minutes rest between sets. All groups were measured before and after the 12-week period with; dynamic leg press and seated-row 6-repetition maximum (6-RM) and functional capacity tests (30-second chair stands and arm curl test, Timed Up and Go), as well as whole-body skeletal muscle mass (SMM) (kg) and fat mass (FM-kg, FM%) assessed by bioelectrical impedance (BIA). Basal blood samples (in the intervention groups only) taken before and after the intervention assessed low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), blood glucose (GLU), triglycerides (TG), high-sensitive C-reactive protein (hsCRP) concentrations and total antioxidant status (TAS) after a 12 h fast. Hormone analysis included prolactin (PRL), progesterone (P) estradiol (ESTR), testosterone (T), follicle stimulating hormone (FSH), and luteinizing hormone (LH). While C showed no changes in any variable, both M and E significantly improved leg press (+ 46 ± 22% and + 21 ± 12%, respectively; p < 0.001) and seated-row (+ 48 ± 21% and + 42 ± 18%, respectively; p < 0.001) 6-RM, as well as all functional capacity outcomes (p < 0.01) due to training. M were the only group to increase muscle mass (+ 3 ± 2%, p < 0.01). Both M and E group significantly (p < 0.05) decreased GLU (–4 ± 6% and –8 ± 10%, respectively), whereas significantly greater decrease was observed in the E compared to the M group (p < 0.05). Only E group significantly decreased TG (–17 ± 25%, p < 0.01), whereas M group increased (+ 15%, p < 0.01). The difference in TG between the groups favored E compared to M group (p < 0.01). These results suggest that short-term “hypertrophic” ST alone mainly improves strength and functional capacity performance, but it influences metabolic and hormonal profile of healthy older women to a lesser extent. In this group of previously untrained older women, time-of-day did not have a major effect on outcome variables, but some evidence suggests that training in the morning may be more beneficial for muscle hypertrophy (i.e. only M significantly increased muscle mass and had larger effect size (M: g = 2 vs. E: g = 0.5).     

A method of isolating viable chondrocytes with proliferative capacity from cryopreserved human articular cartilage

This study aimed to optimise methods of cryopreserving human articular cartilage (AC) tissue for the isolation of late chondrocytes. Human AC specimens from osteoarthritis patients who had undergone total knee replacement were used to optimise the chondrocyte isolation process and the choice of cryoprotective agent (CPA). For AC tissue cryopreservation, intact cored cartilage discs (5 mm diameter) and diced cartilage (0.2–1 mm cubes) from the same sized discs were step cooled and stored in liquid nitrogen for up to 48 h before chondrocyte isolation and in vitro assay of cell viability and proliferative potential. The results showed that 10 % dimethyl sulphoxide in 90 % foetal bovine serum was a successful CPA for chondrocyte cryopreservation. Compared with intact cored discs, dicing of AC tissue into 0.2–1 mm cubes significantly increased the viability and proliferative capacity of surviving chondrocytes after cryopreservation. In situ cross-section imaging using focused ion beam microscopy revealed that dicing of cored AC discs into small cubes reduced the cryo-damage to cartilage tissue matrix. In conclusion, modification of appropriate factors, such as the size of the tissue, cryoprotective agent, and isolation protocol, can allow successful isolation of viable chondrocytes with high proliferative capacity from cryopreserved human articular cartilage tissue. Further studies are required to determine whether these cells may retain cartilage differentiation capacity and provide sufficient chondrocytes for use as implants in clinical applications.