Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing

Objectives: To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. Results: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. Conclusions: This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.     

High-level expression and biochemical characterization of a novel cold-active lipase from <Emphasis Type="Italic">Rhizomucor endophyticus</Emphasis>

Objectives

To identify novel cold-active lipases from fungal sources and improve their production by heterologous expression in Pichia pastoris.

Results

A novel cold-active lipase gene (ReLipB) from Rhizomucor endophyticus was cloned. ReLipB was expressed at a high level in Pichia pastoris using high cell-density fermentation in a 5-l fermentor with the highest lipase activity of 1395 U/ml. The recombinant lipase (RelipB) was purified and biochemically characterized. ReLipB was most active at pH 7.5 and 25 °C. It was stable from pH 4.5–9.0. It exhibited broad substrate specificity towards p-nitrophenyl (pNP) esters (C₂–C₁₆) and triacylglycerols (C₂–C₁₂), showing the highest specific activities towards pNP laurate (231 U/mg) and tricaprylin (1840 U/mg), respectively. In addition, the enzyme displayed excellent stability with high concentrations of organic solvents including cyclohexane, n-hexane, n-heptane, isooctane and petroleum ester and surfactants.

Conclusions

A novel cold-active lipase from Rhizomucor endophyticus was identified, expressed at a high level and biochemically characterized. The high yield and unique enzymatic properties make this lipase of some potential for industrial applications.

     

Production of δ-decalactone from linoleic acid via 13-hydroxy-9(Z)-octadecenoic acid intermediate by one-pot reaction using linoleate 13-hydratase and whole <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cells

Objective

To produce δ-decalactone from linoleic acid by one-pot reaction using linoleate 13-hydratase with supplementation with whole Yarrowia lipolytica cells.

Results

Whole Y. lipolytica cells at 25 g l^?1 produced1.9 g l^?1 δ-decalactone from 7.5 g 13-hydroxy-9(Z)-octadecenoic acid l^?1 at pH 7.5 and 30 °C for 21 h. Linoleate 13-hydratase from Lactobacillus acidophilus at 3.5 g l^?1 with supplementation with 25 g Y. lipolytica cells l^?1 in one pot at 3 h produced 1.9 g l^?1 δ-decalactone from 10 g linoleic acid l^?1 via 13-hydroxy-9(Z)-octadecenoic acid intermediate at pH 7.5 and 30°C after 18 h, with a molar conversion yield of 31 % and productivity of 106 mg l^?1 h^?1.

Conclusion

To the best of our knowledge, this is the first production of δ-decalactone using unsaturated fatty acid.

     

Development of a sensitive molecular detection assay for mango malformation disease caused by <Emphasis Type="Italic">Fusarium mangiferae</Emphasis>

Objectives

To develop a sensitive and specific molecular assay for detection of mango malformation disease (MMD), which is caused primarily by Fusarium mangiferae.

Results

We screened 100 ISSR primers and identified one (UBC888) that directed the stable amplification of a specific gene fragment of 479 bp (GenBank accession number KJ526382). Based on the DNA sequence of this fragment, a pair of SCAR primers (W342 and W1772) were designed to amplify another gene fragment of 1376 bp (GenBank accession number KJ526383), demonstrating the successful conversion of an ISSR marker to a SCAR marker. An effective and simple detection assay for MMD was established based on this pair of PCR primers, with a high level of specificity and sensitivity to the DNA of F. mangiferae and other species of Fusarium both in vitro and in vivo. It can detect as little as 10 pg fungal DNA from the DNA of mango’s tissues.

Conclusions

Our assay provides a practical method for the early diagnosis so that proper prevention of the mango malformation disease can be developed.

     

The Influence of Spatial Attributes on Fragment Occupancy and Population Structure in the Mexican Mantled Howler (<Emphasis Type="Italic">Alouatta palliata mexicana</Emphasis>)

It is essential to document habitat occupancy patterns and population structure to facilitate the survival of primates in areas of anthropogenic disturbance. The overlapping of the Nearctic and Neotropical regions in the Olmec region of Mexico make this area particularly important as part of a natural biological corridor that harbors a high number of endemic species and connects the Atlantic and Pacific coastal plains. We surveyed Alouatta palliata mexicana (Mexican mantled howlers) in a 300-km² area to determine if fragment occupation and subpopulation structure were related to the spatial attributes of the fragments. We measured the fragment size and shape as well as the distances to the nearest road, human settlement, agricultural field, and nearest neighboring fragment. During 1 year (ca. 4500 fieldwork hours) we surveyed 48 fragments, 17 of which were occupied, and counted 198 howlers. Larger fragments that were farther from agricultural activities were more likely to be occupied. Subpopulation size and number of individuals in all age–sex classes increased in larger fragments that were closer to other fragments. We found more females and juveniles, as well as more females per male in fragments that were farther from roads and we found fewer immatures per female, females per male, and individuals per area in more irregular fragments. In addition, more males and immatures per female occurred in fragments that were farther from agricultural fields. The Olmec Region is located at the center of the geographic distribution of mantled howlers in Mexico, and could therefore play a fundamental role in maintaining the contact between different populations. However, our study suggests that mantled howlers are highly threatened by anthropogenic habitat disturbance in this area, mainly through the loss of their habitat and contact with humans.     

Factors Influencing the Survival of Sympatric Gorilla (<Emphasis Type="Italic">Gorilla gorilla gorilla</Emphasis>) and Chimpanzee (<Emphasis Type="Italic">Pan troglodytes troglodytes</Emphasis>) Nests

Accurate and precise surveys of primate abundance provide the basis for understanding species ecology and essential information for conservation assessments. Owing to the elusive nature of wild apes and the vast region of dense forest they inhabit, population estimates of central chimpanzees (Pan troglodytes troglodytes) and western lowland gorillas (Gorilla gorilla gorilla) have largely relied on surveys of their nests. Specific information about the nesting behavior of apes permits the estimation of the number of nests built (nest creation rate). Similarly, information on nest characteristics and environmental factors can be used to estimate the time it takes nests to decay (nest decay rate). Nest creation and decay rates are then used to convert nest density estimates to absolute ape densities. Population estimates that use site-specific estimates of nest creation and decay rates are more accurate and precise. However, it is common practice to generalize these conversion factors across sites because of the additional cost of studies required to gather the information to estimate them. Over a 9-mo study period, we detected and monitored the time to decay of gorilla nests (N = 514) and chimpanzee nests (N = 521) in northern Republic of Congo. We investigated the influence of nest characteristics and environmental factors on nest survivorship and estimated the mean time to nest decay (or equivalently survival) using MARK. Key factors influencing nest decay rate included ape species, forest type, nest height, mean rainfall, nest structure, nest type, and primary aspects of nest construction. Our findings highlight the synergistic effect of behavior and environment on great ape nest degradation, as well as providing practical insights for improving measures to monitor remaining populations of these endangered species.     

Diet and Feeding Behavior of <Emphasis Type="Italic">Leontopithecus chrysomelas</Emphasis> (Callitrichidae) in Degraded Areas of the Atlantic Forest of South-Bahia,Brazil

Habitat disturbance alters plant diversity and food resource availability, affecting the ecology, and ultimately the survival and reproduction, of species depending on those plants. Studies in degraded areas serve to improve our understanding of the consequences of habitat modification for endangered species and to guide conservation actions. We studied diet composition, monthly variation in feeding behavior and fruit feeding time, and dietary diversity in two golden-headed lion tamarin (Leontopithecus chrysomelas) groups ranging in a degraded area of the Atlantic Forest in South-Bahia, Brazil, over a 12-mo period. We recorded feeding behavior and food items consumed through continuous observations and focal animal sampling, and performed dietary analyses on samples from consumed fruits. Substrate manipulation to search for animal prey was the feeding activity most frequently observed, followed by fruit consumption. We observed nectar drinking and exudate feeding at low frequencies from April through July. Bromeliads were the principal foraging substrate. We observed the use of 114 plant species, confirming the large dietary diversity reported for the species. Individual trees from the family Melastomataceae, common in degraded forests, accounted for the highest visiting frequencies (40%). Fruit availability was the main factor explaining variation in monthly fruit feeding time, despite the absence of climatic seasonality. Nutritional or energetic characteristics did not affect fruit choice. Differences in floristic composition appear to be a major determinant of the species’ diet in different study areas. Regional forest restoration programs should consider including advanced forest species, to improve both forest quality and animal mobility between fragments.     

Study of the Serum Copper Levels in Patients with Major Depressive Disorder

Copper may be involved in the pathophysiology of depression. Clinical data on this issue are very limited and not conclusive. The purpose of the study was to determine the copper concentration in the serum of patients with major depressive disorder and to discuss its potential clinical usefulness as a biomarker of the disease. A case–control clinical study included 69 patients with current depressive episode, 45 patients in remission and 50 healthy volunteers. Cu concentration was measured by electrothermal atomic absorption spectrometry (ETAAS). The mean serum copper level in depressed patients was slightly lower (by 11 %; not statistically significant) than in the control group. Furthermore, there was no significant difference in Cu²⁺ concentration between depressive episode and remission, nor between remission and control group. In the remission group were observed significant correlations between copper levels and the average number of relapses over the past years or time of remission. There was no correlation between serum copper and severity of depression, as measured by HDRS and MADRS. The obtained results showed no significant differences between the copper concentration in the blood serum of patients (both with current depressive episode and in remission) and healthy volunteers, as well as the lack of correlations between the copper level in the active stage of the disease and clinical features of the population. Our study is the first conducted on such a large population of patients, so the results may be particularly important and reliable source of knowledge about the potential role of copper in depression.     

Adverse Effects of Ball-Chain Radio-Collars on Female Mantled Howlers (<Emphasis Type="Italic">Alouatta palliata</Emphasis>) in Panama

The placement of radio- and GPS-collars on primates has advanced the fields of primate behavior and ecology and yielded considerable conservation gains. However, to ensure that the placement of tracking collars remains a humane practice, the reporting of both positive and negative outcomes is required. Here, we evaluate the impacts of radio-collars on the reproduction, behavior, and health of female mantled howlers (Alouatta palliata) on Barro Colorado Island, Panama. In June 2005, as part of a behavioral study, we fitted 16 female howlers with ball-chain radio-collars weighing 1.2% of the mean adult female weight. We monitored these 16 collared females and 64 noncollared females across an annual cycle, and recaptured the collared females in July 2006 for collar removal. The mortality rate for collared females across the study year was 6.3% (N=1), while the net loss of noncollared females from study groups was 15.6% (N=10). All collared females reproduced. The behaviors of collared and noncollared females were not significantly different. However, on recapture to remove all remaining collars (N = 13), 38% of recaptured collared females presented with damage to the dermal layer on the back of the neck and 31% presented with more severe damage extending into the subcutaneous tissue and muscle. Given this physical damage, which was not apparent through binoculars, we cannot recommend using ball-chain radio-collars on female mantled howlers without careful welfare monitoring.