Multifunction of autophagy-related genes in filamentous fungi

Autophagy (macroautophagy), a highly conserved eukaryotic mechanism, is a non-selective degradation process, helping to maintain a balance between the synthesis, degradation and subsequent recycling of macromolecules to overcome various stress conditions. The term autophagy denotes any cellular process which involves the delivery of cytoplasmic material to the lysosome for degradation. Autophagy, in filamentous fungi plays a critical role during cellular development and pathogenicity. Autophagy, like the mitogen-activated protein (MAP) kinase cascade and nutrient-sensing cyclic AMP (cAMP) pathway, is also an important process for appressorium turgor accumulation in order to penetrate the leaf surface of host plant and destroy the plant defense. Yeast, an autophagy model, has been used to compare the multi-valued functions of ATG (autophagy-related genes) in different filamentous fungi. The autophagy machinery in both yeast and filamentous fungi is controlled by Tor kinase and both contain two distinct phosphatidylinositol 3-kinase complexes. In this review, we focus on the functions of ATG genes during pathogenic development in filamentous fungi.     

Root colonization and growth promotion of sunflower (Helianthus annuus L.) by phosphate solubilizing Enterobacter sp. Fs-11

An Enterobacter sp. Fs-11 was isolated from sunflower rhizosphere, identified on the basis of 16S rRNA gene sequence analysis (GeneBank accession no. GQ179978) and studied for its root colonization and growth promotion ability in sunflower. Morphologically, it was rod shaped Gram-negative, motile bacterium, producing 4.5 μg mL(-1) indole acetic acid in tryptophan-supplemented medium. It utilized 27 out of 95 substrates in BIOLOG GN2 micro plate system. It was able to convert insoluble tri-calcium phosphate to soluble phosphorus up to 43.5 μg mL(-1) with decrease in pH of the medium up to 4.5 after 10 days incubation at 28 ± 2 °C in the Pikovskaya's broth. High performance liquid chromatography of cell free supernatant showed that Fs-11 produced malic acid and gluconic acid (2.43 and 16.64 μg mL(-1), respectively) in Pikovskaya's broth. Analysis of 900 bp fragment of pyrroloquinoline quinine pqqE gene sequence showed 98 % homology with that of E. cloacae pqqE gene. Confocal laser scanning microscope revealed strong colonization of fluorescently labeled Fs-11 with sunflower roots. Sunflower inoculation with Fs-11 and its rifampicin resistant derivative in sterile sand and natural soil showed that Fs-11 colonized sunflower roots up to 30 days after transplanting in both sterile sand as well as natural soil. Moreover, Fs-11 inoculation resulted in increased plant height, fresh weight, dry weight and total phosphorus contents as compared to un-inoculated plants. The data showed that Enterobacter sp. Fs-11 is an efficient phosphate solubilizing and plant growth promoting rhizobacterium and has great potential to be used as bio-inoculant for sunflower under phosphorus deficient conditions.     

AChE inhibitor: a regio- and stereo-selective 1,3-dipolar cycloaddition for the synthesis of novel substituted 5,6-dimethoxy spiro[5.3']-oxindole-spiro-[6.3″]-2,3-dihydro-1H-inden-1″-one-7-(substituted aryl)-tetrahydro-1H-pyrrolo[1,2-c][1,3]thiazole

Pyrrolothiazolyloxindole analogues share vital pharmacological properties, considered useful in Alzheimer's disease (AD). The aim of this study was synthesis and evaluate pyralothiazolyloxindole analogues if possess acetyl cholinesterase (AChE) inhibitory activity. The easily accessible one-pot synthesis of these compounds resulted to be significantly less difficult and expensive than that of donepezil. Several compounds possess anti-cholinesterase activity in the order of micro and sub-micromolar. Particularly, compound was the most potent inhibitors of the series against acetyl cholinesterase enzyme with IC(50) 0.11μmol/L.     

Origin of immunoglobulin isotype switching

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Highlights? Switching in sharks consistent with immunization and cytidine deaminase expression ? Isotype switching can be reciprocal between genes of established positions ? Switch joints show DNA double-strand breakage repaired by nonhomologous end-joining ? Unlike in tetrapods, switching in shark is bidirection between IgM and IgW     

Vitamin D levels in asymptomatic adults--a population survey in Karachi, Pakistan

Background

It is well established that low levels of 25(OH) Vitamin D (<30 ng/dL) are a common finding world over, affecting over a billion of the global population. Our primary objective was to determine the prevalence of vitamin D deficiency and insufficiency in the asymptomatic adult population of Karachi, Pakistan and the demographic, nutritional and co-morbidity characteristics associated with serum vitamin D levels.

Methods

A cross-sectional population survey was conducted at two spaced out densely populated areas of the city. Serum levels of 25OHVitamin D were measured and GFR as renal function was assessed by using 4 variable MDRD formula.

Results

Our sample of 300 had a median age of 48(interquartile range 38–55) years. The median level of serum vitamin D was 18.8 (IQ range 12.65–24.62) ng/dL. A total of 253 (84.3%) respondents had low levels (<30 ng/dL) of 25OH vitamin D. Serum PTH and vitamin D were negatively correlated (r = −0.176, p = 0.001). The median PTH in the vitamin D sufficiency group was 38.4(IQ range28.0–48.8)pg/mL compared with 44.4(IQ range34.3–56.8) pg/mL in the deficiency group (p = 0.011).The median serum calcium level in the sample was 9.46(IQ range 9.18–9.68) ng/dL. Low serum levels of vitamin D were not associated with hypertension (p = 0.771) or with an elevated spot blood pressure (p = 0.164).In our sample 75(26%) respondents had an eGFR corresponding to stage 2 and stage 3 CKD. There was no significant correlation between levels of vitamin D and eGFR (r = −0.127, p-value = 0.277).Respondents using daily vitamin D supplements had higher 25 OH vitamin D levels (p-value = 0.021).

Conclusion

We observed a high proportion of the asymptomatic adult population having low levels of vitamin D and subclinical deterioration of eGFR. The specific cause(s) for this observed high prevalence of low 25OH vitamin D levels are not clear and need to be investigated further upon.     

High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting

The ability to track microbes in real time in vivo is of enormous value for preclinical investigations in infectious disease or gene therapy research. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumours following systemic administration. Bioluminescent Imaging (BLI) represents a powerful tool for use with bacteria engineered to express reporter genes such as lux. BLI is traditionally used as a 2D modality resulting in images that are limited in their ability to anatomically locate cell populations. Use of 3D diffuse optical tomography can localize the signals but still need to be combined with an anatomical imaging modality like micro-Computed Tomography (μCT) for interpretation.In this study, the non-pathogenic commensal bacteria E. coli K-12 MG1655 and Bifidobacterium breve UCC2003, or Salmonella Typhimurium SL7207 each expressing the luxABCDE operon were intravenously (i.v.) administered to mice bearing subcutaneous (s.c) FLuc-expressing xenograft tumours. Bacterial lux signal was detected specifically in tumours of mice post i.v.-administration and bioluminescence correlated with the numbers of bacteria recovered from tissue. Through whole body imaging for both lux and FLuc, bacteria and tumour cells were co-localised. 3D BLI and μCT image analysis revealed a pattern of multiple clusters of bacteria within tumours. Investigation of spatial resolution of 3D optical imaging was supported by ex vivo histological analyses. In vivo imaging of orally-administered commensal bacteria in the gastrointestinal tract (GIT) was also achieved using 3D BLI. This study demonstrates for the first time the potential to simultaneously image multiple BLI reporter genes three dimensionally in vivo using approaches that provide unique information on spatial locations.     

Evaluation of portable point-of-care CD4 counter with high sensitivity for detecting patients eligible for antiretroviral therapy

Background

Accurate, inexpensive point-of-care CD4+ T cell testing technologies are needed that can deliver CD4+ T cell results at lower level health centers or community outreach voluntary counseling and testing. We sought to evaluate a point-of-care CD4+ T cell counter, the Pima CD4 Test System, a portable, battery-operated bench-top instrument that is designed to use finger stick blood samples suitable for field use in conjunction with rapid HIV testing.

Methods

Duplicate measurements were performed on both capillary and venous samples using Pima CD4 analyzers, compared to the BD FACSCalibur (reference method). The mean bias was estimated by paired Student''s t-test. Bland Altman plots were used to assess agreement.

Results

206 participants were enrolled with a median CD4 count of 396 (range; 18–1500). The finger stick PIMA had a mean bias of −66.3 cells/µL (95%CI −83.4−49.2, P<0.001) compared to the FACSCalibur; the bias was smaller at lower CD4 counts (0–250 cells/µL) with a mean bias of −10.8 (95%CI −27.3−+5.6, P = 0.198), and much greater at higher CD4 cell counts (>500 cells/µL) with a mean bias of −120.6 (95%CI −162.8, −78.4, P<0.001). The sensitivity (95%CI) of the Pima CD4 analyzer was 96.3% (79.1–99.8%) for a <250 cells/ul cut-off with a negative predictive value of 99.2% (95.1–99.9%).

Conclusions

The Pima CD4 finger stick test is an easy-to-use, portable, relatively fast device to test CD4+ T cell counts in the field. Issues of negatively-biased CD4 cell counts especially at higher absolute numbers will limit its utility for longitudinal immunologic response to ART. The high sensitivity and negative predictive value of the test makes it an attractive option for field use to identify patients eligible for ART, thus potentially reducing delays in linkage to care and ART initiation.     

Recognizing and managing the expanded risk of tumor lysis syndrome in hematologic and solid malignancies

Background

Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34⁺ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters.

Results

By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34⁺ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34⁺ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-X _L and BCLW. In contrast, pro-apoptotic BID and BIM _EL expression were downregulated in ET leukocytes. Increased BCL-X_L protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly.

Conclusions

Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.