A novel procedure for protein extraction from formalin-fixed paraffin-embedded tissues

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases.     

Steady-state kinetics of the plasma membrane H+-ATPase from red beet (Beta vulgaris): its inhibition by vanadate and inorganic phosphate

The intial velocity vs ATP concentration curves obtained with the plasma membrane H⁺-ATPase from red beet ( Beta vulgaris L.) did not follow classical Michaelis-Menten kinetics. A rate equation containing second-order terms in ATP concentration in both the numerator and the denominator was used to obtain a significantly better fit to the data. The observed deviations from Michaelis-Menten kinetics were more pronounced in the presence of potassium ions. The inhibition caused by inorganic phosphate was partial. i.e. the ATPase activity extrapolated at an infinite phosphate concentration was not zero. In contrast, the inhibition produced by orthovanadate was nearly total. The inhibitions caused by both phosphate and vanadate were uncompetitive with respect to ATP and enhanced by potassium ions and high concentrations of dimethyl sulfoxide. a solvent used to lower the water activity of the reaction medium. The ATP-dependent proton transport was stimulated by potassium ions and was inhibited by phosphate only at high ATP concentrations. A kinetic mechanism, in which the H⁺-ATPase can adopt two conformations during its catalytic cycle and can form a ternary enzyme-ATP-phosphate complex able to hydrolyze bound ATP. is proposed to explain those results.     

Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici

Sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. In plants, recent evidence suggests that their metabolic precursors, the long-chain bases (LCBs) act as bioactive molecules in the immune response. Interestingly, the virulence of two unrelated necrotrophic fungi, Fusarium verticillioides and Alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of sphinganine-analog mycotoxins (SAMs). These metabolites inhibit de novo synthesis of sphingolipids in their hosts causing accumulation of LCBs, which are key regulators of programmed cell death. Therefore, to gain more insight into the role of sphingolipids in plant immunity against SAM-producing necrotrophic fungi, we disrupted sphingolipid metabolism in Nicotiana benthamiana through virus-induced gene silencing (VIGS) of the serine palmitoyltransfersase (SPT). This enzyme catalyzes the first reaction in LCB synthesis. VIGS of SPT profoundly affected N. benthamiana development as well as LCB composition of sphingolipids. While total levels of phytosphingosine decreased, sphinganine and sphingosine levels increased in SPT-silenced plants, compared with control plants. Plant immunity was also affected as silenced plants accumulated salicylic acid (SA), constitutively expressed the SA-inducible NbPR-1 gene and showed increased susceptibility to the necrotroph A. alternata f. sp. lycopersici. In contrast, expression of NbPR-2 and NbPR-3 genes was delayed in silenced plants upon fungal infection. Our results strongly suggest that LCBs modulate the SA-dependent responses and provide a working model of the potential role of SAMs from necrotrophic fungi to disrupt the plant host response to foster colonization.     

S-layer proteins of Lactobacillus acidophilus inhibits JUNV infection

We investigated the regulation of Hsp27 phosphorylation by protein kinase C δ (PKCδ) during etoposide-induced apoptosis. The phosphorylation of Hsp27 at Ser78 was temporally correlated with the proteolytic activation of PKCδ during apoptosis. Hsp27 phosphorylation was dependent on the activity of PKCδ since treatment with rottlerin, a chemical inhibitor of PKCδ, or overexpression of a PKCδ dominant negative mutant abolished the phosphorylation. In addition, recombinant PKCδ phosphorylated Hsp27 at Ser78 in vitro. Moreover, caspase-3 was specifically activated following Hsp27 phosphorylation at Ser78. Pull-down assays using a phosphomimetic Hsp27 mutant revealed that binding between Hsp27 and cytochrome c was abolished by the phosphorylation. These results suggest that Hsp27 dissociates from cytochrome c following PKCδ-mediated phosphorylation at Ser78, which allows formation of the apoptosome and stimulates apoptotic progression.     

Results of the Implementation of a Pilot Model for the Bidirectional Screening and Joint Management of Patients with Pulmonary Tuberculosis and Diabetes Mellitus in Mexico

Background

Recently, the World Health Organisation and the International Union Against Tuberculosis and Lung Disease published a Collaborative Framework for the Care and Control of Tuberculosis (TB) and Diabetes (DM) (CFTB/DM) proposing bidirectional screening and joint management.

Objective

To evaluate the feasibility and effectiveness of the CFTB/DM in Mexico. Design. Prospective observational cohort. Setting. 15 primary care units in 5 states in Mexico. Participants: Patients aged ≥20 years diagnosed with DM or pulmonary TB who sought care at participating clinics. Intervention: The WHO/Union CFTB/DM was adapted and implemented according to official Mexican guidelines. We recruited participants from July 2012 to April 2013 and followed up until March 2014. Bidirectional screening was performed. Patients diagnosed with TB and DM were invited to receive TB treatment under joint management. Main outcome measures. Diagnoses of TB among DM, of DM among TB, and treatment outcomes among patients with DM and TB.

Results

Of 783 DM patients, 11 (1.4%) were unaware of their TB. Of 361 TB patients, 16 (4.4%) were unaware of their DM. 95 TB/DM patients accepted to be treated under joint management, of whom 85 (89.5%) successfully completed treatment. Multiple linear regression analysis with change in HbA1c and random capillary glucose as dependent variables revealed significant decrease with time (regression coefficients (β)  = −0.660, (95% confidence interval (CI), −0.96 to −0.35); and β = −1.889 (95% CI, −2.77 to −1.01, respectively)) adjusting by sex, age and having been treated for a previous TB episode. Patients treated under joint management were more likely to experience treatment success than patients treated under routine DM and TB programs as compared to historical (adjusted OR (aOR), 2.8, 95%CI 1.28–6.13) and same period (aOR 2.37, 95% CI 1.13–4.96) comparison groups.

Conclusions

Joint management of TB and DM is feasible and appears to improve clinical outcomes.     

A1298C polymorphism of the MTHFR gene and neural tube defects in the state of Yucatan, Mexico

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR) of the folate metabolism pathway is a candidate gene for neural tube defects (NTDs). Frequency of the second common polymorphism, A1298C, in the MTHFR gene is not well known in Mexico. Conflicting results exist regarding the association of A1298C-MTHFR with NTDs. One explanation for this controversy might be that alleles are differently distributed among various populations. The aim of the study was to determine the frequency of the A1298C-MTHFR polymorphism and its association with NTDs in a population of Yucatan, Mexico. METHODS: Genotyping was performed by use of polymerase chain reaction with restriction fragment length polymorphisms using MbOII endonuclease (PCR-RFLPs MbOII). Allele and genotype frequencies were compared between cases with NTDs, their mothers and fathers with matched controls based on an association analysis using EpiInfo software. RESULTS: A1298C genotypes were distributed according to Hardy-Weinberg expectations for all studied groups. Frequencies of allele C and heterozygous AC genotype were significantly higher in males (p = .006 and p = .011, respectively) in control group. Significant differences were not observed between cases and controls, except in mothers of NTD cases compared with mothers of healthy offspring for both allele C and heterozygous AC genotype (p = .009 and p = .01, respectively). CONCLUSIONS: The polymorphism A1298C-MTHFR is not associated with NTDs, except for mothers, suggesting only a maternal association with having NTD-affected offspring in the Yucatan population. The frequency of allele C in the control population was 10%, which is significantly lower than in other reported control populations worldwide (p < .01).     

Effect of plasticity in hatching on duration as a precompetent swimming larva in the nudibranch Phestilla sibogae

Abstract. Plasticity in hatching can balance risks of benthic and pelagic development and thereby affect the extent of larval dispersal. Veligers of the nudibranch Phestilla sibogae hatched from their individual capsules if the encapsulated embryos were scattered from a torn gelatinous egg ribbon. Hatching occurred as early as day 4 at 23°–25°C. The early hatchlings lacked a propodium, swam, and were not yet competent to settle and metamorphose. Hatching may be induced by predation: crabs consumed egg ribbons, and a portunid crab, caught in the act of tearing an egg ribbon, scattered encapsulated embryos. Undisturbed egg masses hatched as late as 9–11 d at 23°–25°C, or as early as 8 d in a trial at 26°C. Late hatchlings had a well-developed propodium, and 20–100% metamorphosed within a day of exposure to the inducer from the nudibranch's coral prey. A few metamorphosed nudibranchs were found within hatching egg masses. Thus, the veligers can hatch so late that many are competent to metamorphose or so early that the obligate planktonic period can last 4 or more days. An attack by a predator means the benthic habitat is dangerous for the embryos, and swimming is presumably the safer option. In the absence of disturbance, the veligers hatch when ready or nearly ready to settle.     

Rhizosphere bacteria of maize with chitinolytic activity and its potential in the control of phytopathogenic fungi

The chitinase enzyme was identified in isolated bacteria of maize rhizosphere as well as its potential for the biological control of fungi associated at seeds of the same plant. The production of chitinase enzyme was found in the genera identified as Acinetobacter, Bacterium, Burkholderia, Paenibacillus, Pseudomonas, Rhizobium, Shewanella, Sphingomonas and Stenotrophomonas. Bacterial isolates with ability to degrade fungal mycelium from maize fungi as Fusarium and Alternaria among others, were detected. Bacterial chitinase activity and the presence of the chiA gene were determined. The inoculation of chitinolytic bacteria showed a positive effect in the control of fungi in maize seeds. The results support the potential use of chitinase enzyme producing bacteria on the control of phytopathogenic fungi.