Design and use of group-specific primers and probes for real-time quantitative PCR

Real-time quantitative polymerase chain reaction (qPCR) has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil, water, sediments, and sludge. Although qPCR is a very useful technique for nucleic acid quantification, accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used. Many aspects of conducting qPCR assays have become increasingly routine and automated; however, one of the most important aspects, designing and selecting primer and probe sets, is often a somewhat arcane process. In many cases, failed or non-specific amplification can be attributed to improperly designed primer-probe sets. This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays. We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes. qPCR assays using group-specific primers and probes designed with this method, have been used to successfully quantify 16S ribosomal Ribonucleic Acid (16S rRNA) gene copy numbers from target methanogenic and ammonia-oxidizing bacteria in various laboratory- and full-scale biologic processes. Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.     

Characteristics of fungal flora in onion farmlands with potential link to human mycotic keratitis

Acquisition of mycotic keratitis has been linked to agricultural activities. Although fungi were identified in injured eyes, data derived directly from farmlands are limited. Following five cases of fungal corneal ulcers in onion harvesters in the monsoon area of Taiwan, the present study further determined fungi genera and their concentrations in onion crops and surface soils of onion farmlands. In total, 52, 100 and 100 samples of soils, onion leaves and bulb scales, respectively, were collected from three monsoon and one non-monsoon farmlands. Fungal colonies were counted and microscopically examined after incubation at 25°C for 4–7 days on malt extract agar with 40?g?mL^?1 chloramphenicol. Results showed the peak fungi levels were mostly observed at harvest time. Among keratitis-related fungi, Aspergillus predominated in soils as well as in onion bulb scales, while Alternaria in onion leaves. Cladosporium was also prevalent in soils, onion leaves and scales. Other keratitis-related fungi included Acremonium, Curvalaria, Fusarium, Mycelia, Penicillium, Rhizoctonia and Rhizopus. The present study provides an exposure link between onion harvesters and presence of keratitis-related fungi from onion crops and soils, which may account for mycotic ocular infection via hand-to-eye contact and airborne transmission during the monsoon season.     

Effects of cyanobacterium Microcystis aeruginosa on the filtration rate and mortality of the freshwater bivalve Corbicula leana

We compared filtering rates (FR) and mortalities between freshwater filter-feeding bivalve Corbicula leana acclimatized (AM) and non-acclimatized (NAM) to an cyanobacterial bloom (mainly Microcystis aeruginosa) over seven days. Both mussel populations were simultaneously stocked into mesocosms constructed in littoral zones of a eutrophic lake at a density of 740 ind. m(-2) for 16 days. NAMs decreased the concentration of chlorophyll-a 50% less than AMs. For the first seven days, the FRs (0.46-0.61) and mortality rate (slope = -30.2, r = -0.95) of NAMs were higher than those of AMs, possibly due to a sudden increase in unselective filtering. From the eighth day NAM mortality decreased rapidly and then stabilized, becoming similar to that of AMs through the end of the experiment. Stocking both AMs and NAMs increased the NH4-N concentration in the water, and the mortality rates of both mussel populations were correlated with NH4-N. In contrast, other nutrients and microcystin concentrations were not significantly associated with mussel mortality. These results indicate that although a sudden introduction of non-acclimatized C. leana may partially control phytoplankton biomass fora short period, previous short-term acclimatization is needed to minimize mussel mortality.     

A critical exploration of blood and environmental chromium concentration among oral cancer patients in an oral cancer prevalent area of Taiwan

The growing incidence of oral cancer (OC) in Taiwan has become a crucial public health concern. In particular, Changhua, a county in central Taiwan, carries persistently high OC incidence rate, with an alarmingly high male/female ratio of OC incidence. Previous epidemiological studies had found that the incidence is spatially correlated with the level of soil content to certain heavy metals in the central Taiwan area. Soil and the human body both intake environmental heavy metals, which can be absorbed through various ways. The soil metal concentration is an index of possible environmental exposure to heavy metal, and the blood metal concentration somewhat reflects the level of the exposure on the human body. Metallic carcinogen is likely to generate free radicals and play a role in many cancers, and many studies had reported that environmental exposure to heavy metals is an important risk factor for developing cancer. Studies on animals showed that chronic intake of chromium (Cr) could induce OC. This study aims to explore the association between the Cr concentration in the farm soil and in the blood of OC patients. We recruited 79 OC patients from Changhua County, with their lifestyle being adjusted in regression analysis. The results showed that the Cr concentration in the blood of OC patients is significantly higher than the background value, and is positively associated with the Cr concentration in the soil surrounding their residence (p-value < 0.023). Because Changhua County is only with moderate prevalence of the known OC habitual risk factors, an environmental factor related to heavy metal Cr exposure is suspected. Future investigations may verify the causal relation between Cr and OC.     

Effects of sedimentation on the gorgonian <Emphasis Type="Italic">Subergorgia suberosa</Emphasis> (Pallas, 1766)

Effects of oceanic and terrestrial sediment suspensions on the behavioral traits activity, colony expansion, and mucus formation of the gorgonian octocoral Subergorgia suberosa (Pallas, 1766) were studied in the laboratory. Increased sediment concentration significantly changed the logits of activity indices (as colony expansion) and was correlated with decreased activity. For both sources of sediments, the activity index decreased as the concentration of sediment increased. The level of decrease was more significance for terrestrial sediments. The activity index showed no clear time pattern for the control group, but a decrease over time occurred in both sediment treatment groups. Activity change was linearly correlated with exposure time and concentration of terrestrial and ocean sediments. The activity indices of polyps were negatively correlated with the formation of mucus sheets (correlation = −0.536, p = 0.048). For both sources of sediments, the frequency of mucus sheet formation (MSF) increased with sediment concentration. Increases in terrestrial sediment loads resulted in more striking effects. The probability for MSF under different sediment groups at different concentrations over time was modeled by regressions. Frequency of polyp activity is suggested as a suitable biomarker to monitor the effects of sedimentation stress.     

A CFD study of the transport and fate of airborne droplets in a ventilated office: The role of droplet−droplet interactions

• Coulomb and Lennard−Jones forces were considered for droplet interactions. • The net droplet interactions were repulsive. • Repulsive droplet interactions increased the transport of droplets. • Repulsive droplet interactions significantly modified the fate of droplets. Previous studies reported that specially designed ventilation systems provide good air quality and safe environment by removing airborne droplets that contain viruses expelled by infected people. These water droplets can be stable in the environment and remain suspended in air for prolonged periods. Encounters between droplets may occur and droplet interactions should be considered. However, the previous studies focused on other physical phenomena (air flow, drag force, evaporation) for droplet transport and neglected droplet interactions. In this work, we used computational fluid dynamics (CFD) to simulate the transport and fate of airborne droplets expelled by an asymptomatic person and considered droplet interactions. Droplet drag with turbulence for prediction of transport and fate of droplets indicated that the turbulence increased the transport of 1 μm droplets, whereas it decreased the transport of 50 μm droplets. In contrast to only considering drag and turbulence, consideration of droplet interactions tended to increase both the transport and fate. Although the length scale of the office is much larger than the droplet sizes, the droplet interactions, which occurred at the initial stages of release when droplet separation distances were shorter, had a significant effect in droplet fate by considerably manipulating the final locations on surfaces where droplets adhered. Therefore, it is proposed that when an exact prediction of transport and fate is required, especially for high droplet concentrations, the effects of droplet interactions should not be ignored.     

Distribution and early life history of <Emphasis Type="Italic">Kaupichthys</Emphasis> leptocephali (family Chlopsidae) in the central Indonesian Seas

Leptocephali of the widely distributed tropical marine eels of the genus Kaupichthys (family Chlopsidae) were collected around Sulawesi Island during a sampling survey in the Indonesian Seas in late September and early October 2002, and the otolith microstructure of 24 of the 59 specimens captured was examined to learn about the larval growth rates and spawning times of these small sized eels. Leptocephali ranging in size from 25 to 60 mm were collected in Makassar Strait and the Celebes Sea, but they were most abundant in the semi-enclosed Tomini Bay of northeast Sulawesi Island. The Kaupichthys leptocephali examined had 39–161 otolith growth increments. Their back-calculated hatching dates indicated that five age groups were present and each group appeared to have been spawned around the full moon of previous months. Average growth rate estimates of the first two age groups were 0.65 and 0.54 mm/day for the 27.4–30.4 and 37.6–45.6 mm age classes. The growth rates of the oldest three age groups (52.0–60.8 mm) appeared to have slowed down after they reached their approximate maximum size. An increase in increment widths at the outer margin of the otoliths of those larger than 53 mm suggested that the process of metamorphosis had begun even though there were few external morphological changes indicating metamorphosis. It is hypothesized that chlopsid leptocephali have an unusually short gut that may not need to move forward during early metamorphosis. The presence of four age classes in Tomini Bay suggests that the Togian Islands region may be productive habitats for Kaupichthys juveniles and adults.     

An individual-based model to study the reproduction of egg bearing copepods: Application to Eurytemora affinis (Copepoda Calanoida) from the Seine estuary,France

Limited empirical studies have elucidated the daily egg production and associated reproductive processes of egg bearing copepod. Herein, we present an individual-based model which constitutes a realistic representation of the reproduction in egg bearing copepods. The model has been parameterized using an extensive set of experimental data obtained from the literature and from the laboratory and field experiments on the estuarine copepod Eurytemora affinis. The proposed model takes into account the adult female longevity, the clutch size and interclutch duration, which is a function of egg maturation time and latency time required by the female after egg hatching to produce a new clutch. The embryonic development time and hatching success are also taken into account. The effect of temperature on the means and variances of above-mentioned reproductive parameters has been also incorporated. A multi agent system based generic platform “Mobidyc” has been used to generate and calibrate the model. The model demonstrates the reproductive parameters of females of E. affinis which is validated through individual based experiments. Temperature specific simulations provide a dynamical explanation of temperature effect on the cumulative egg production. The daily survival principally affects the number of clutches produced per female during its life span. The results obtained in the present study by combining temperature and survival effects reveal the relatively greater importance of the first factor on the daily egg production of egg-carrying copepods. The present model is generic and hence easily applicable to other animals with comparable reproductive strategy.     

Recovery of solid fuel from municipal solid waste by hydrothermal treatment using subcritical water

Hydrothermal treatments using subcritical water (HTSW) such as that at 234 °C and 3 MPa (LT condition) and 295 °C and 8 MPa (HT condition) were investigated to recover solid fuel from municipal solid waste (MSW). Printing paper, dog food (DF), wooden chopsticks, and mixed plastic film and sheets of polyethylene, polypropylene, and polystyrene were prepared as model MSW components, in which polyvinylchloride (PVC) powder and sodium chloride were used to simulate Cl sources.While more than 75% of carbon in paper, DF, and wood was recovered as char under both LT and HT conditions, plastics did not degrade under either LT or HT conditions. The heating value (HV) of obtained char was 13,886-27,544 kJ/kg and was comparable to that of brown coal and lignite. Higher formation of fixed carbon and greater oxygen dissociation during HTSW were thought to improve the HV of char.Cl atoms added as PVC powder and sodium chloride to raw material remained in char after HTSW. However, most Cl originating from PVC was found to converse into soluble Cl compounds during HTSW under the HT condition and could be removed by washing.From these results, the merit of HTSW as a method of recovering solid fuel from MSW is considered to produce char with minimal carbon loss without a drying process prior to HTSW. In addition, Cl originating from PVC decomposes into soluble Cl compound under the HT condition. The combination of HTSW under the HT condition and char washing might improve the quality of char as alternative fuel.     

Effects of fine particulate matter and its constituents on emergency room visits for asthma in southern Taiwan during 2008–2010: a population-based study

Environmental Science and Pollution Research - This population-based study evaluated the short-term association between fine particulate matter (PM2.5) concentrations and its constituents and...