Nested distributions of bat flies (Diptera: Streblidae) on Neotropical bats: artifact and specificity in host-parasite studies

We examined the structure of ectoparasitic bat fly infestations on 31 well‐sampled bat species, representing 4 Neotropical families. Sample sizes varied from 22 to 1057 bats per species, and bat species were infested by 4 to 27 bat fly species. Individual bats supported smaller infracommunities (the set of parasites co‐occurring on an individual host), ranging from 1 to 5 fly species in size, and no bat species had more than 6 bat fly species characteristically associated with it (its primary fly species). Nestedness analyses used system temperature (BINMATNEST algorithm) because it is particularly well‐suited for analysis of interaction networks, where parasite records may be nested among hosts and host individuals simultaneously nested among parasites. Most species exhibited very low system temperatures (mean 3.14°; range 0.14–12.28°). Simulations showed that nested structure for all 31 species was significantly stronger than simulated values under 2 of the 3 null hypotheses, and about half the species were also nested under the more stringent conditions of the third null hypothesis. Yet this structure disappears when analyses are restricted to “primary” associations of fly species (flies on their customary host species), which exclude records thought to be atypical, transient, or potential contaminants. Despite comprising a small fraction of total parasite records, such anomalies represent a considerable part of the statistical state‐space, offering the illusion of significant ecological structure. Only well understood and well documented systems can make distinctions between primary and other occurrence records. Generally, nestedness appears best developed in host‐parasite systems where infestations are long‐term and accumulate over time. Dynamic, short‐term infestations by highly mobile parasites like bat flies may appear to be nested, but such structure is better understood in terms of host specificity and accidental occurrences than in terms of prevalence, persistence, or hierarchical niche relations of the flies.     

Mutation discovery in the mouse using genetically guided array capture and resequencing

Forward genetics (phenotype-driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between observable phenotype and causative genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next-generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long-range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array-based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (~200 kb) from each of five noncomplementing Kit mutants (one known allele and four unknown alleles) and have successfully identified and validated a nonsynonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover causative mutations. Importantly, these data also provide a specific methodological foundation for the development of large-scale mutation detection efforts in the laboratory mouse. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. M. D’Ascenzo and C. Meacham contributed equally to this work.     

Elevated endogenous testosterone concentrations potentiate muscle androgen receptor responses to resistance exercise

The purpose of this study was to determine the influence of endogenous circulating testosterone (T) on muscle androgen receptor (AR) responses to acute resistance exercise (RE). Six healthy men (26 ± 4 years; 176 ± 5 cm; 75.8 ± 11.4 kg) performed a knee extension exercise protocol on two occasions separated by 1–3 weeks. Rest preceded one trial (i.e., control [CON] trial) and a high-volume upper-body RE protocol designed to increase circulating T preceded the other trial (i.e., high T [HT] trial). Serial blood samples were obtained throughout each trial to determine circulating T concentrations. Biopsies of the vastus lateralis were obtained pre-RE (REST), 10-min post-RE (+10), and 180-min post-RE (+180) to determine muscle AR content. Circulating T concentrations remained stable during CON. Alternately, HT significantly (p ≤ 0.05) increased T concentrations above resting values (+16%). Testosterone area-under-the-time curve during HT exceeded CON by 14%. AR content remained stable from REST to +10 in both trials. Compared to the corresponding +10 value, muscle AR content at +180 tended to decrease during CON (−33%; p = 0.10) but remained stable during HT (+40%; p = 0.17). Muscle AR content at +180 during the HT trial exceeded the corresponding CON value. In conclusion, acute elevations in circulating T potentiated muscle AR content following RE.     

Synthesis and evaluation of novel tricyclic benzo[4.5]cyclohepta[1.2]pyridine derivatives as NMDA/NR2B antagonists

A novel series of annulated tricyclic compounds was synthesized and evaluated as NMDA/NR2B antagonists. Structure–activity development was directed towards in vitro optimization of NR2B activity and selectivity over the hERG K⁺ channel. Preferred compounds were subsequently evaluated for selectivity in an α₁-adrenergic receptor binding counter-screen and a cell-based assay of NR2B activity.     

Prevalence of Antibiotic Resistance in Drinking Water Treatment and Distribution Systems

The occurrence and spread of antibiotic-resistant bacteria (ARB) are pressing public health problems worldwide, and aquatic ecosystems are a recognized reservoir for ARB. We used culture-dependent methods and quantitative molecular techniques to detect and quantify ARB and antibiotic resistance genes (ARGs) in source waters, drinking water treatment plants, and tap water from several cities in Michigan and Ohio. We found ARGs and heterotrophic ARB in all finished water and tap water tested, although the amounts were small. The quantities of most ARGs were greater in tap water than in finished water and source water. In general, the levels of bacteria were higher in source water than in tap water, and the levels of ARB were higher in tap water than in finished water, indicating that there was regrowth of bacteria in drinking water distribution systems. Elevated resistance to some antibiotics was observed during water treatment and in tap water. Water treatment might increase the antibiotic resistance of surviving bacteria, and water distribution systems may serve as an important reservoir for the spread of antibiotic resistance to opportunistic pathogens.The occurrence and spread of antibiotic-resistant bacteria (ARB) are pressing public health problems worldwide, and aquatic ecosystems are a recognized reservoir for ARB and antibiotic resistance genes (ARGs) (4, 6, 8, 11, 12, 15, 39). Naturally occurring ARB and ARGs in the aquatic environment are selected for and enriched for by antibiotics found in sewage and agricultural runoff, which result from the widespread and increased use of antibiotics (4, 11, 12, 15, 38). Historically, concerns about the microbial quality of drinking water have focused on the occurrence of pathogens in drinking water distribution systems (5, 34). However, the presence of trace levels of antibiotics and ARB in source water and finished drinking water may also greatly affect public health and is an emerging issue for the general public and the drinking water industry (3, 30). Although several studies have detected ARB in drinking water systems (2, 3, 20, 30, 38), most previous studies focused on cultivable bacteria and/or indicator organisms. Little is known about the fate of ARGs in drinking water systems, and it was recently proposed that ARGs are emerging contaminants (24).We used culture-dependent methods and molecular techniques to investigate the prevalence and dynamics of heterotrophic ARB and ARGs in a drinking water source (source RW-P) and treated drinking water (source DW-P) (see Materials and Methods in the supplemental material). We tested water from a drinking water plant located in Michigan and tap water from several small cities located in Michigan and Ohio (sources TW-1, TW-2, TW-3, and TW-4). Two independent samples were collected each time at each collection site at three different times, and we used four replicates from each sample for tests. We tested bacterial resistance to the following antibiotics: amoxicillin (amoxicilline), chloramphenicol, ciprofloxacin, gentamicin, rifampin (rifampicin), sulfisoxazole, and tetracycline. We also examined the presence of eight ARGs, including beta-lactam resistance genes (bla_TEM and bla_SHV), chloramphenicol resistance genes (cat and cmr), sulfonamide resistance genes (sulI and sulII), and tetracycline resistance genes (tetO and tetW).Total heterotrophic plate counts (HPC) were determined using R₂A agar without added antibiotics. The water treatment process reduced the total HPC from 9.9 × 10⁶ CFU/100 ml in source water to 68 CFU/100 ml in treated drinking water, indicating that there was efficient removal and/or deactivation of total HPC (Table ​(Table1).1). In contrast, the total 16S rRNA gene copy number decreased from 3.4 × 10⁷ copies/100 ml in source water to 1.6 × 10⁶ copies/100 ml in treated drinking water (Fig. ​(Fig.1).1). The discrepancy between the reduction in the HPC and the reduction in the total 16S rRNA gene copy number suggests that the final disinfection step effectively inactivated bacteria but most of the dead or damaged cells were still present in finished drinking water. The number of HPC in tap water ranged from 3.44 × 10² to 6.1 × 10⁴ CFU/100 ml water, values that are lower than those for source water but significantly higher than those for treated drinking water, indicating that there is regrowth of bacteria in drinking water distribution systems. The copy numbers of total 16S rRNA genes in tap water ranged from 2.45 × 10⁵ to 1.02 × 10⁷ copies/100 ml water. The higher levels suggested by the 16S rRNA data are consistent with results of previous studies demonstrating that only 5 to 10% and 1% of bacteria in wastewater and soil, respectively, can be cultivated or identified by culture-based methods (9, 37). A significant correlation (P < 0.05, R² = 0.78) was found between the 16S rRNA gene copy number and the total HPC if treated drinking water (DW-P) data were not included (Fig. ​(Fig.1).1). This suggests that cultivable bacteria in drinking water represent only a small portion of the total bacterial biomass. Including treated drinking water (DW-P) data resulted in a distorted correlation, suggesting that a large proportion of the 16S rRNA genes present came from dead and/or damaged cells. The levels of total heterotrophic bacteria were significantly higher in tap water (TW-1) than in treated drinking water (DW-P), indicating that there was bacterial regrowth in the water distribution system.Open in a separate windowFIG. 1.Heterotrophic bacteria and the 16S rRNA gene in different water samples. (A) Copy numbers of the 16S rRNA gene and numbers of heterotrophic bacteria (CFU) in 100 ml water. (B) Correlation (P < 0.05, R² = 0.78) between the copy number of the 16S rRNA gene and the number of heterotrophic bacteria in different water samples (without the data for DW-P). RW-P, source water from the drinking water treatment plant; DW-P, finished drinking water from the drinking water treatment plant; TW-1, tap water from the city where the drinking water treatment plant is located; TW-2, TW-3, and TW-4, tap water from three towns in Michigan and Ohio close to the city where the TW-1 drinking water treatment plant is located. The statistical analysis was done using six samples for each type of water sample. Lg, log₁₀.

TABLE 1.

Prevalence of ARB HPC in source water, finished drinking water, and tap water from four towns^a

Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV³⁵⁶) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV³⁵⁶ neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.The mammalian high-temperature requirement A (HtrA) family of serine proteases is defined by a characteristic trypsin-like serine protease domain and one or two C-terminal PDZ domains. Four mammalian HtrA proteins have been identified to date, HtrA1–4. HtrA1 (also called PRSS11) is a ubiquitously expressed extracellular serine protease which contains a signal sequence for secretion, an insulin-like growth factor (IGF)²-binding protein domain, and a Kazal-type serine protease inhibitor domain in addition to the serine protease domain and one C-terminal PDZ domain (1). HtrA1 has been implicated in the progression of several pathologies including age-related macular degeneration, cancer, Alzheimer disease, rheumatoid arthritis, and osteoarthritis (OA) (2–10). HtrA1 has also been shown to inhibit osteoblast mineralization (11).Expression of HtrA1 has been found to be elevated in articular cartilage in association with OA (5). In addition, HtrA1 levels are up-regulated in murine cartilage after experimentally induced joint damage (6). The physiological role of HtrA1 in OA disease progression as well as in other pathologies is unclear. Preliminary studies using in vitro digestion assays suggest that HtrA1 might be capable of digesting cartilage extracellular matrix (ECM) proteins such as fibromodulin, cartilage oligomeric matrix protein (COMP), fibronectin, decorin, and aggrecan (6, 12, 13). Furthermore, it was recently reported that elevated levels of HtrA1 protein (∼7-fold above normal) are present in synovial fluids obtained from OA patients and that fibronectin fragments generated by HtrA1 cleavage induced the expression of catabolic enzymes such as matrix metalloproteinases-1 (MMP-1) and MMP-3 in synovial fibroblasts (4). HtrA1 has also been shown to modulate multiple signaling pathways in vitro. It binds to transforming growth factor-β family proteins including transforming growth factor-β1 and bone morphogenetic proteins 2 and 4 and inhibits signaling mediated by these factors (14, 15). In addition, HtrA1 has been shown to cleave IGF-binding protein-5 and possibly regulate signaling mediated by IGF (16). These findings suggest that the protease HtrA1 may play a physiological role in cartilage during OA.Articular cartilage is made up of chondrocytes surrounded by the ECM comprised mainly of the proteoglycan, aggrecan, and type II collagen. During normal homeostasis there is a dynamic balance between anabolic activities such as proteoglycan synthesis as well as catabolic activities in which the ECM is destroyed. When the catabolic activities of proteases, such as MMPs and aggrecanases, offset new matrix synthesis, focal degradation and loss of articular cartilage occurs, resulting in the development of OA. In some in vitro digestion studies, we and others have shown degradation of aggrecan by recombinant HtrA1 (6, 12, 13). In the present study we set out to examine the physiological relevance of aggrecan cleavage by HtrA1 in OA disease progression.     

Early soft rot colonization of Scots sapwood pine in above-ground exposure

The early colonization of Scots pine (Pinus sylvestris L.) sapwood exposed above ground (staple bed) was studied. Two different types of exposures were used, one in an open field and the other in a shaded field. Decay type and degree of degradation due to soft rot, and mass and strength loss of wood were correlated. Fungal species in Scots pine sapwood were identified by sequencing, using the fungal nuclear ribosomal DNA (nrDNA) after 24 months.The most abundant decay type found was soft rot, which also agreed with the mass loss (7–8%). Pine sapwood did not differ significantly between the two sites regarding the average mass loss during the time of exposure. The early colonization of wood by soft rot fungi together with mass loss indicates that this fungal type might be more common in above-ground conditions than recognized earlier.     

Mechanical Restoration of California Mixed‐Conifer Forests: Does it Matter Which Trees Are Cut?

The montane ecosystems of northern California have been subjected to repeated manipulation and active fire suppression for over a century, resulting in changes in community structure that contribute to increased wildfire hazard. Ecosystem restoration via reduction of stand density for wildfire hazard mitigation has received substantial attention in recent years; however, many ecological questions remain unanswered. This study compares belowground effects of two alternative forest thinning treatments designed to restore the large, old tree component of late‐seral structure, one of which focuses on restoring Pinus ponderosa dominance (Pine‐preference) and the other of which promotes development of large trees regardless of species (Size‐preference). We evaluated forest floor and soil chemical and microbial parameters in six experimental thinning treatment units of 40 ha each in the Klamath National Forest of northern California 5–6 years after thinning. Inorganic N availability, soil organic C content, phenol oxidase activity, and forest floor C:N ratio were greater in the Size‐preference treatment, whereas forest floor N and soil pH were greater in the Pine‐preference treatment. Our results indicate that these two thinning strategies produce differences in the soil environment that has the potential to affect growth rates of trees that remain, as well as the growth and survivorship of newly established seedlings. Thus, which species/individuals are removed during structural restoration of these mixed‐conifer forests matters both to the belowground components of the ecosystem today and the vegetation and productivity of the ecosystem in future decades.