NMR assignment of a KlbA intein precursor from Methanococcus jannaschii

The backbone and side chain resonance assignments of a precursor of the KlbA intein from Methanococcus jannaschii have been determined, based on triple-resonance experiments with the uniformly [¹³C,¹⁵N]-labeled protein.     

Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method.     

A database of mutants and effects of site-directed mutagenesis experiments on G protein-coupled receptors

A database system and computer programs for storage and retrieval of information about guanine nucleotide-binding protein (G protein) -coupled receptor mutants and associated biological effects have been developed. Mutation data on the receptors were collected from the literature and a database of mutants and effects of mutations was developed. The G protein-coupled receptor, family A, point mutation database (GRAP) provides detailed information on ligand-binding and signal transduction properties of more than 2130 receptor mutants. The amino acid sequences of receptors for which mutation experiments have been reported were aligned, and from this alignment mutation data may be retrieved. Alternatively, a search form allowing detailed specification of which mutants to retrieve may be used, for example, to search for specific amino acid substitutions, substitutions in specific protein domains or reported biological effects. Furthermore, ligand and bibliographic oriented queries may be performed. GRAP is available on the Internet (URL: http://www-grap.fagmed.uit.no/GRAP/homepage.html ) using the World-Wide Web system. © 1996 Wiley-Liss, Inc.     

Effects of preconception lifestyle intervention in infertile women with obesity: The FIT-PLESE randomized controlled trial

BackgroundWomen with obesity and infertility are counseled to lose weight prior to conception and infertility treatment to improve pregnancy rates and birth outcomes, although confirmatory evidence from randomized trials is lacking. We assessed whether a preconception intensive lifestyle intervention with acute weight loss is superior to a weight neutral intervention at achieving a healthy live birth.Methods and findingsIn this open-label, randomized controlled study (FIT-PLESE), 379 women with obesity (BMI ≥ 30 kg/m²) and unexplained infertility were randomly assigned in a 1:1 ratio to 2 preconception lifestyle modification groups lasting 16 weeks, between July 2015 and July 2018 (final follow-up September 2019) followed by infertility therapy. The primary outcome was the healthy live birth (term infant of normal weight without major anomalies) incidence. This was conducted at 9 academic health centers across the United States. The intensive group underwent increased physical activity and weight loss (target 7%) through meal replacements and medication (Orlistat) compared to a standard group with increased physical activity alone without weight loss. This was followed by standardized empiric infertility treatment consisting of 3 cycles of ovarian stimulation/intrauterine insemination. Outcomes of any resulting pregnancy were tracked. Among 191 women randomized to standard lifestyle group, 40 dropped out of the study before conception; among 188 women randomized to intensive lifestyle group, 31 dropped out of the study before conception. All the randomized women were included in the intent-to-treat analysis for primary outcome of a healthy live birth. There were no significant differences in the incidence of healthy live births [standard 29/191(15.2%), intensive 23/188(12.2%), rate ratio 0.81 (0.48 to 1.34), P = 0.40]. Intensive had significant weight loss compared to standard (−6.6 ± 5.4% versus −0.3 ± 3.2%, P < 0.001). There were improvements in metabolic health, including a marked decrease in incidence of the metabolic syndrome (baseline to 16 weeks: standard: 53.6% to 49.4%, intensive 52.8% to 32.2%, P = 0.003). Gastrointestinal side effects were significantly more common in intensive. There was a higher, but nonsignificant, first trimester pregnancy loss in the intensive group (33.3% versus 23.7% in standard, 95% rate ratio 1.40, 95% confidence interval [CI]: 0.79 to 2.50). The main limitations of the study are the limited power of the study to detect rare complications and the design difficulty in finding an adequate time matched control intervention, as the standard exercise intervention may have potentially been helpful or harmful.ConclusionsA preconception intensive lifestyle intervention for weight loss did not improve fertility or birth outcomes compared to an exercise intervention without targeted weight loss. Improvement in metabolic health may not translate into improved female fecundity.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02432209","term_id":"NCT02432209"}}NCT02432209.

Richard Legro and colleagues investigate the impact of a preconception weight loss intervention on healthy live birth rates in women with obesity and unexplained infertility.     

Malnutrition,anemia, micronutrient deficiency and parasitic infections among schoolchildren in rural Tanzania

BackgroundMalnutrition, anemia, micronutrient deficiency and parasitic infections continue to impact the nutritional status and health of children in lower-income countries. However, not enough data concerning this issue is available. The aim of this study was to assess the distribution of nutritional indicators, anemia and micronutrient deficiency and their underlying risk factors among schoolchildren in south-eastern Tanzania.Methodology/Principal findingsThis cross-sectional study enrolled primary schoolchildren aged 6–12 years from Kikwawila and Kiberege wards, Tanzania. In total, 471 schoolchildren underwent a physical examination and provided blood, stool and urine samples for an assessment of the levels of different micronutrients, nutritional and anemia status, and parasitic infection status. We employed bivariate and multivariate logistic regression to determine the association between nutritional statuses, anemia, micronutrient deficiency and parasitic infections. We found that 23.90%, 12.60% and 16.20% of schoolchildren were stunted, underweight and wasted, respectively. About 14.0% of schoolchildren were found to be anemic. Children diagnosed with Plasmodium falciparum infection were more likely to have low levels of ferritin (aOR: 10.40, 95% CI: 2.88-40.53) and elevated levels of serum soluble transferrin receptor (aOR: 3.59, 95% CI: 1.27-11.23), respectively. Vitamin A (34.71%) and vitamin B12 (8.79%) were the most prevalent micronutrients found to be deficient in diagnosed children. Finally, we found that schoolchildren attending the most rural schools were five times more likely to be diagnosed with at least one micronutrient deficiency (aOR: 5.04, 95% CI: 2.38–11.44).Conclusions/SignificanceMalnutrition, anemia and micronutrient deficiency still pose a significant health burden among schoolchildren living in rural Tanzania. To effectively tackle this burden, health interventions such as deworming, micronutrient supplementation, vector control, health education and access to clean water and improved sanitation should be strengthened and made sustainable.     

The Availability of Phosphorus and the Species Composition of the Spring Phytoplankton in Lake Erken

The spring development of phytoplankton and the phosphorus content were monitored intensively in Laken Erken during the springs of 1979 and 1980. The dominant species were dinoflagellates, mainly Woloszynskia ordinata and Peridinium aciculiferum, during the entire spring of 1979. The long period of ice-cover (21 weeks) favoured the motile species, which were able to utilize the available light immediately below the ice. A considerable vertical migration was noticed in March 1979. These large and slow-growing algae were able to store luxury phosphorus, and surplus phosphorus concentrations up to 12 μg P/l were measured, which corresponded to an algal content of 10 μg P/mg C. These values were more than twice as high as those recorded earlier in Lake Erken. Although a spring outburst of diatoms did not occur in 1979 and 1980, the algae were phosphorus-limited in May, as indicated by high alkaline phosphatase activity, low algal surplus phosphorus content and high inorganic N : P ratios.     

Impact of cell culture process changes on endogenous retrovirus expression

Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other process changes may be unnecessary.     

Spatial ecology of wolverines at the southern periphery of distribution

Wolverines (Gulo gulo) in the conterminous United States have experienced range contraction, are uncommon, and have been designated as warranted for protection under the United States Endangered Species Act. Data from the southern edge of the wolverine's circumpolar distribution is sparse, and development of effective conservation strategies would benefit from a more complete understanding of the species' ecology. We captured and radio-monitored 30 wolverines in the Greater Yellowstone Ecosystem (GYE), tested for seasonal habitat selection by elevation band, and examined a suite of spatial characteristics to clarify our understanding of the wolverine's niche. Wolverines in GYE selected for areas >2,600 m latitude-adjusted elevation (LAE; n = 2,257 wolverine locations [12 F, 6 M]). Wolverines avoided areas <2,150 m LAE, including during winter when the vast majority of ungulates are pushed to these elevations by deep snow. Wolverine home ranges were large relative to body size, averaging 303 km² for adult females and 797 km² for adult males (n = 13 [8 F, 5 M] and 33 wolverine-years). Resident adults fit with Global Positioning System (GPS) collars used an area >75% the size of their multi-year home range in an average of 32 days (n = 7 [5 F, 2 M]). Average movement rates of 1.3 km/2-hr indicated that both sexes move distances equivalent to the diameter of their home range every 2 days or the circumference of their home range in <1 week (n = 1,329 2-hr movements, n = 12 individuals [7 F, 5 M]). This capability for movement, the short time-frame over which home ranges were developed, and a lack of home range overlap by same sex adults ( , 90% CI = 0.0–4.8%, n = 22 pairs) suggested territoriality. We estimated wolverine density to be 3.5/1,000 km² of area >2,150 m LAE (95% CI = 2.8–9.6). Dispersal movements extended to at least 170 km for both sexes (n = 5 F, 2 M). At the southern edge of distribution, where suitable and unsuitable conditions exist in close proximity, wolverines selected high-elevation areas near alpine tree-line where a mix of forest, meadow, and boulder fields were present, deep snow-cover existed during winter, and low temperatures near freezing can occur throughout the year. Persistence in these areas where the growing season is brief requires large home ranges that are regularly patrolled, a social system that provides exclusive access to resources, and low densities. These characteristics, along with low reproductive rates, are prevalent throughout the species range, indicating that wolverines are specialists at exploiting a cold, unproductive niche where interspecific competition is limited. The vulnerability inherent in occupying this unproductive niche was likely influential in previous declines within the conterminous United States and will remain a factor as wolverines encounter modern human influences. Conserving wolverines in the conterminous United States will require collaborative management over a large geographic scale. © 2011 The Wildlife Society.     

Repeated trans-watershed hybridization among haplochromine cichlids (Cichlidae) was triggered by Neogene landscape evolution

The megadiverse haplochromine cichlid radiations of the East African lakes, famous examples of explosive speciation and adaptive radiation, are according to recent studies, introgressed by different riverine lineages. This study is based on the first comprehensive mitochondrial and nuclear DNA dataset from extensive sampling of riverine haplochromine cichlids. It includes species from the lower River Congo and Angolan (River Kwanza) drainages. Reconstruction of phylogenetic hypotheses revealed the paradox of clearly discordant phylogenetic signals. Closely related mtDNA haplotypes are distributed thousands of kilometres apart and across major African watersheds, whereas some neighbouring species carry drastically divergent mtDNA haplotypes. At shallow and deep phylogenetic layers, strong signals of hybridization are attributed to the complex Late Miocene/Early Pliocene palaeohistory of African rivers. Hybridization of multiple lineages across changing watersheds shaped each of the major haplochromine radiations in lakes Tanganyika, Victoria, Malawi and the Kalahari Palaeolakes, as well as a miniature species flock in the Congo basin (River Fwa). On the basis of our results, introgression occurred not only on a spatially restricted scale, but massively over almost the whole range of the haplochromine distribution. This provides an alternative view on the origin and exceptional high diversity of this enigmatic vertebrate group.     

Thrombospondin-2 and SPARC/osteonectin are critical regulators of bone remodeling

Thrombospondin-2 (TSP2) and osteonectin/BM-40/SPARC are matricellular proteins that are highly expressed by bone cells. Mice deficient in either of these proteins show phenotypic alterations in the skeleton, and these phenotypes are most pronounced under conditions of altered bone remodeling. For example, TSP2-null mice have higher cortical bone volume and are resistant to bone loss associated with ovariectomy, whereas SPARC-null mice have decreased trabecular bone volume and fail to demonstrate an increase in bone mineral density in response to a bone-anabolic parathyroid hormone treatment regimen. In vitro, marrow stromal cell (MSC) osteoprogenitors from TSP2-null mice have increased proliferation but delayed formation of mineralized matrix. Similarly, in cultures of SPARC-null MSCs, osteoblastic differentiation and mineralized matrix formation are decreased. Overall, both TSP2 and SPARC positively influence osteoblastic differentiation. Intriguingly, both of these matricellular proteins appear to impact MSC fate through mechanisms that could involve the Notch signaling system. This review provides an overview of the role of TSP2 and SPARC in regulating bone structure, function, and remodeling, as determined by both in vitro and in vivo studies.