A Phloxine-Azure-Hematoxylin Sequence for Differential Staining of Cells in Pancreatic Islets

Sections of 6 μ from tissues fixed in Susa or in Bouin's fluid (without acetic acid) and embedded in paraffin were attached to slides with Mayer's albumen, dried at 37 C for 12 hr, deparaffinized and hydrated. The sections fixed in Susa were transferred to a I₂-K₁ solution (1:2:300 ml of water); rinsed in water, decolorized in 5% Na₂S₂O₃; washed in running water, and rinsed in distilled water. Those fixed in Bouin's were transferred to 80% alcohol until decolorized, then rinsed in distilled water. All sections were stained in 1% aqueous phloxine, 10 min; rinsed in distilled water and transferred to 3% aqueous phosphotungstic acid, 1 min; rinsed in distilled water; stained 0.5 min in 0.05 azure II (Merck), washed in water; and finally, nuclear staining in Weigert's hematoxylin for 1 min was followed by a rinse in distilled water, rapid dehydration through alcohols, clearing in xylene and covering in balsam or a synthetic resin. In the completed stain, islet cells appear as follows: A cells, purple; B cells, weakly violet-blue; D cells, light blue with evident granules; exocrine cells, grayish blue with red granules.     

Intraspecific Variation in Chemical Attraction of Rice to Insect Predators

The olfactory response of predators of the brown planthopper,Nilaparvata lugensStål, to different genotypes of rice (14 cultivars and breeding lines ofOryza sativaL. and 1 wild species,Oryza nivaraSharma et Shastry) was measured in an airflow olfactometer. Odor from rice plants attracted more females of the mirid predatorCyrtorhinus lividipennisReuter than plain air (control) on only 6 of the 15 rice genotypes. Orientation ofC. lividipennistoward volatiles of certain rice genotypes was apparent even when the plants were free of the brown planthopper. However, the predator distinguished between prey-infested and uninfested plants and preferred plants with eggs over plants with nymphs. The predator did not distinguish different stages of plant growth (vegetative, booting, or flowering). Plants artificially injured to simulate brown planthopper oviposition wounds were not as attractive to the predator as plants on which the planthopper had oviposited. The preassay preconditioning on the cultivar TN1 did not produce a predator bias for this genotype. This suggests that rearing effects or chemically mediated associative learning reported for some natural enemies did not influenceC. lividipennis'host response. Results with another predator, the coccinellidMicraspis hirashimaiSasaji, produced less consistent behavior. Planthopper-infested plants attracted more females ofM. hirashimaithan unifested plants in only 1 of the 12 rice genotypes evaluated. Implications for augmenting predators by rice cultivar selection and modification are discussed.     

Nitrogen-fixing cyanobacteria as source of phycobiliprotein pigments. Composition and growth performance of ten filamentous heterocystous strains

Ten strains of filamentous, heterocystous nitrogen-fixing blue-green algae (cyanobacteria) were screened for growth performance and tolerance to temperature, pH, irradiance and salinity, together with their potential as producers of phycobiliprotein pigments. Phycobiliproteins typically accounted for about 50% total cell protein, the prevalent type being C-phycocyanin, followed by alloppycocyanin, with levels of 17 and 11% d.wt, respectively, in some strains of Anabaena and Nostoc. C-phycoerythrin was the major pigment in several Nostoc strains, reaching 10% d.wt. Some strains represent, therefore, excellent sources of one or more phycobiliproteins. All strains tolerated an irradiance of ca 2000 μmol photon m^-2 s^-1. Anabaena sp. ATCC 33047 and Nostoc sp. (Albufera) exhibited the widest optimum range of both temperature (30–45 and 25–40 °C) and pH (6.5–9.5 and 6.0–9.0) for growth, the former also showing significant salt tolerance. In an outdoor open system, productivity of cultures of two phycoerythrin-rich strains of Nostoc was over 20 g (d.wt) m^-2 d^-1 during summer. The growth performance of the allophycocyanin-rich Anabaena sp. ATCC 33047 in outdoor semi-continuous culture has been assessed throughout the year. Productivity values under optimized conditions ranged from 9 (winter) to 24 (summer) g (d.wt) m^-2 d^-1.     

Helping decision making for reliable and cost‐effective 2b‐RAD sequencing and genotyping analyses in non‐model species

High‐throughput sequencing has revolutionized population and conservation genetics. RAD sequencing methods, such as 2b‐RAD, can be used on species lacking a reference genome. However, transferring protocols across taxa can potentially lead to poor results. We tested two different IIB enzymes (AlfI and CspCI) on two species with different genome sizes (the loggerhead turtle Caretta caretta and the sharpsnout seabream Diplodus puntazzo) to build a set of guidelines to improve 2b‐RAD protocols on non‐model organisms while optimising costs. Good results were obtained even with degraded samples, showing the value of 2b‐RAD in studies with poor DNA quality. However, library quality was found to be a critical parameter on the number of reads and loci obtained for genotyping. Resampling analyses with different number of reads per individual showed a trade‐off between number of loci and number of reads per sample. The resulting accumulation curves can be used as a tool to calculate the number of sequences per individual needed to reach a mean depth ≥20 reads to acquire good genotyping results. Finally, we demonstrated that selective‐base ligation does not affect genomic differentiation between individuals, indicating that this technique can be used in species with large genome sizes to adjust the number of loci to the study scope, to reduce sequencing costs and to maintain suitable sequencing depth for a reliable genotyping without compromising the results. Here, we provide a set of guidelines to improve 2b‐RAD protocols on non‐model organisms with different genome sizes, helping decision‐making for a reliable and cost‐effective genotyping.     

Increased dosage of Ink4/Arf protects against glucose intolerance and insulin resistance associated with aging

Recent genome‐wide association studies have linked type‐2 diabetes mellitus to a genomic region in chromosome 9p21 near the Ink4/Arf locus, which encodes tumor suppressors that are up‐regulated in a variety of mammalian organs during aging. However, it is unclear whether the susceptibility to type‐2 diabetes is associated with altered expression of the Ink4/Arf locus. In the present study, we investigated the role of Ink4/Arf in age‐dependent alterations of insulin and glucose homeostasis using Super‐Ink4/Arf mice which bear an extra copy of the entire Ink4/Arf locus. We find that, in contrast to age‐matched wild‐type controls, Super‐Ink4/Arf mice do not develop glucose intolerance with aging. Insulin tolerance tests demonstrated increased insulin sensitivity in Super‐Ink4/Arf compared with wild‐type mice, which was accompanied by higher activation of the insulin receptor substrate (IRS)‐PI3K‐AKT pathway in liver, skeletal muscle and heart. Glucose uptake studies in Super‐Ink4/Arf mice showed a tendency toward increased ¹⁸F‐fluorodeoxyglucose uptake in skeletal muscle compared with wild‐type mice (P = 0.079). Furthermore, a positive correlation between glucose uptake and baseline glucose levels was observed in Super‐Ink4/Arf mice (P < 0.008) but not in wild‐type mice. Our studies reveal a protective role of the Ink4/Arf locus against the development of age‐dependent insulin resistance and glucose intolerance.     

A population-based study of first and second-line drug-resistant tuberculosis in a high-burden area of the Mexico/United States border

The resistance of 139 Mycobacterium tuberculosis (MTB) isolates from the city of Monterrey, Northeast Mexico, to first and second-line anti-TB drugs was analysed. A total of 73 isolates were susceptible and 66 were resistant to anti-TB drugs. Monoresistance to streptomycin, isoniazid (INH) and ethambutol was observed in 29 cases. Resistance to INH was found in 52 cases and in 29 cases INH resistance was combined with resistance to two or three drugs. A total of 24 isolates were multidrug-resistant (MDR) resistant to at least INH and rifampicin and 11 MDR cases were resistant to five drugs. The proportion of MDR-TB among new TB cases in our target population was 0.72% (1/139 cases). The proportion of MDR-TB among previously treated cases was 25.18% (35/139 cases). The 13 polyresistant and 24 MDR isolates were assayed against the following seven second-line drugs: amikacin (AMK), kanamycin (KAN), capreomycin (CAP), clofazimine (CLF), ethionamide (ETH), ofloxacin (OFL) and cycloserine (CLS). Resistance to CLF, OFL or CLS was not observed. Resistance was detected to ETH (10.80%) and to AMK (2.70%), KAN (2.70%) and CAP (2.70%). One isolate of MDR with primary resistance was also resistant to three second-line drugs. Monterrey has a high prevalence of MDR-TB among previously treated cases and extensively drug-resistant-MTB strains may soon appear.     

In vitro assessment of the acaricidal activity of computer-selected analogues of carvacrol and salicylic acid on Rhipicephalus (Boophilus) microplus

Rhipicephalus (Boophilus) microplus is a tick that causes huge economic losses in cattle. The indiscriminate use of acaricides has generated resistance to most compounds present on the market. This makes further investigation on other potential acaricides necessary, the in silico assay being an alternative to the design of new compounds. In the present study a biosilico assay was performed using TOMOCOMD-CARDD (TOpological MOlecular COMputer Design-Computer-Aided Rational Drug Design) and WEKA (Waikato Environment for Knowledge Analysis) software. Two carvacrol and four salicylic acid derivatives, synthesized by conventional methods and evaluated with the larval packet test on larvae of R. (B.) microplus were selected. All evaluated compounds presented acaricidal activity; however, ethyl 2-methoxybenzoate (91.8 ± 1.7 % mortality) and ethyl 2,5-dihydroxybenzoate (89.1 ± 1.6 % mortality) showed greater activity than salicylic acid. With regard to the carvacrol analogues, carvacrol acetate (67.8 ± 2.1 % mortality) and carvacrol methyl ether (71.7 ± 1.6 % mortality) also showed greater activity than carvacrol (35.9 ± 3.2 % mortality). TOMOCOMD-CARDD and WEKA software were helpful tools in the search for alternative structures with potential acaricidal activity on R. (B.) microplus.     

Pollen biology in four Mediterranean Quercus species

Potential pollen production, viability and germination were studied in the most important species of Quercus in the mountains of Córdoba to determine the contribution of each species to the total amount of airborne pollen. The results were compared over two consecutive years with different rainfall patterns. The viability of pollen grains was determined at anther opening, and during the pollination period, in order to determine potential pollination capacity. Results indicated that there were differences in the number of pollen grains produced by stamen in the four species. Equally, there were differences in the number of flowers among the species, being Q. suber the species with higher number of catkins groups and flowers per individual tree. Total pollen production per tree can be summarized in the following proportion: 1: 3: 3: 6 (Q. coccifera, Q. ilex ssp. ballota, Q. faginea and Q. suber). Potential pollen viability was estimated using the Fluorochromatic Reaction (FCR) and a germination assay. The results have shown that Quercus potential pollen viability is high and declines slowly with time. Q. coccifera was the species with the highest percentage of germination, with Q. suber being the lowest.