Isolation of adipose tissue mesenchymal stem cells without tissue destruction: A non-enzymatic method

The conventional enzymatic method is widely used for mesenchymal stem cells (MSCs) isolation from adipose tissue. The method holds major drawbacks; it is costly, time-consuming and results in a heterogeneous cell population. Besides, digestion of extracellular matrix causes cell injury and compromise proliferation and differentiation of the cells. Also, because of over handling the samples are also prone to contamination. Here, we introduce a non-enzymatic method for MSCs isolation without disturbing the cells habitat. Small pieces of adipose tissue obtained from animal or human liposuction were explanted into a culture flask, immobilized by fetal bovine serum (FBS) and incubated overnight. The explants were then irrigated with DMEM containing FBS. Within few days, the fibroblast-like cells migrated from the tissue and proliferated rapidly. When subconfluent, the cells were harvested, expanded through 3 passages and used for immunophenotyping and differentiation assays. As judged by flow cytometric analysis of surface markers (CD44⁺, CD105⁺, CD34⁻, CD45⁻), Oil Red O and Alizarin Red staining, the MSCs isolated by our non-enzymatic method were pluripotent and exhibited the potential for differentiation into adipocyte and osteoblast. Great isolation yields, homogeneity of isolated cells, brief procedure, and high economy are the advantages of our method over the conventional protocol.     

Involvement of the cynABDS operon and the CO2-concentrating mechanism in the light-dependent transport and metabolism of cyanate by cyanobacteria

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO-) to CO2 and NH3 through the action of a cytosolic cyanase which required HCO3- as a second substrate. The ability to metabolize NCO- relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO2-concentrating mechanism (CCM), and light. Inactivation of cynS, encoding cyanase, and cynA yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO2, HCO3-, NO3-, NO2-, Cl-, PO4(2-), and SO4(2-) did not contribute to the cellular flux of NCO- and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO- transporter. In the S. elongatus strain PCC7942 DeltachpX DeltachpY mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (Ci) (HCO3- + CO2) pool. Unlike wild-type cells, the rate of NCO- decomposition by the DeltachpX DeltachpY mutant was severely depressed at low external Ci concentrations, indicating that the CCM was essential in providing HCO3- for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO- and Ci. Putative cynABDS operons were identified in the genomes of diverse Proteobacteria, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.     

Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.     

Molecular evidence for association of Cotton leaf curl Alabad virus with yellow vein mosaic disease of okra in North India

Two virus isolates (OY77 and OY81B) from okra plants showing yellow vein mosaic, downward curling and vein twisting symptoms were collected from different farmer's fields in Karnal, Haryana state, India. The genomes of the two isolates were amplified, cloned, sequenced and analysed. The analysis indicated that the isolates are similar with 89.2% nucleotide sequence identity. Based on the current threshold cut-off value for taxonomy distinguishing the genus begomoviruses species from strains, the two isolates are designated as strains of Cotton leaf curl Alabad virus (CLCuAV) which shared nucleotide sequence identity of >90% with CLCuAV infecting cotton in Pakistan. Phylogenetic and recombination analyses of the major genome component of OY77 and OY81B is derived from different begomviruses (CLCuAV, BYVMV, CLCuMuV) as the foremost parents for evolution of these new recombinant strains.     

Assessment of left ventricular diastolic suction in dogs using wave-intensity analysis

Two apparently different types of mechanisms have emerged to explain diastolic suction (DS), that property of the left ventricle (LV) that tends to cause it to refill itself during early diastole independent of any force from the left atrium (LA). By means of the first mechanism, DS depends on decreased elastance [e.g., the relaxation time constant (tau)] and, by the second, end-systolic volume (V(LVES)). We used wave-intensity analysis (WIA) to measure the total energy transported by the backward expansion wave (I(W-)) during LV relaxation in an attempt to reconcile these mechanisms. In six anesthetized, open-chest dogs, we measured aortic, LV (P(LV)), LA (P(LA)), and pericardial pressures and LV volume by orthogonal ultrasonic crystals. Mitral velocity was measured by Doppler echocardiography, and aortic velocity was measured by an ultrasonic flow probe. Heart rate was controlled by pacing, V(LVES) by volume loading, and tau by isoproterenol or esmolol administration. I(W-) was found to be inversely related to tau and V(LVES). Our measure of DS, the energy remaining after mitral valve opening, I(W-DS), was also found to be inversely related to tau and V(LVES) and was approximately 10% of the total "aspirating" energy generated by LV relaxation (i.e., I(W-)). The size of the Doppler (early filling) E wave depended on I(W-DS) in addition to I(W+), the energy associated with LA decompression. We conclude that the energy of the backward-going wave generated by the LV during relaxation depends on both the rate at which elastance decreases (i.e., tau) and V(LVES). WIA provides a new approach for assessing DS and reconciles those two previously proposed mechanisms. The E wave depends on DS in addition to LA decompression.     

The positive effects of Mediterranean-neutropenic diet on nutritional status of acute myeloid leukemia patients under chemotherapy

Introduction

Cancer and chemotherapy-induced malnutrition increase death, reduce the response to treatment, and increase multiple kinds of side effects. The aim of this study was to investigate the effects of Mediterranean-neutropenic diet on the nutritional status of acute myeloid leukemia patients under chemotherapy.

Materials and Methods

50 patients were divided into two groups by a random allocation scheme: the Mediterranean-neutropenic diet (n = 25) and the neutropenic diet group (n = 25). The intervention was implemented during a one month period. The nutritional status was evaluated based on PG-SGA. Serum albumin levels and dietary intake were also measured.

Results

After the intervention, the mean serum albumin level in the intervention group was significantly higher than the beginning of the study (p = 0.09) and in comparison with the control group (p = 0.01). Also, the mean serum albumin level in the control group significantly decreased at the end of the study compared to the beginning of the study (p = 0.03). After intervention, the nutritional status of the patients in the intervention group was significantly improved compared to the control group.

Conclusion

In general, based on the results of this study, the Mediterranean neutropenic diet improves nutritional status during chemotherapy by increasing food intake, preventing weight loss and increasing serum albumin levels

     

Threatening biomarkers in lupus pregnancy: Biochemistry and genetic challenges

Objective

Using genetic markers and miRs work strongly beside other sensitive biomarkers in lupus management during sensitive period of pregnancy.

Methods

PubMed and Google Scholar databases were searched from 2000 to 2017 using the terms “lupus,” “lupus pregnancy,” “biomarkers,” “micro-RNA,” “polymorphisms,” “anti-phospholipid antibodies,” and “cluster differentiation markers.”

Discussion

Complement is a valuable biomarker in lupus pregnancy. However, the complement profile has ambiguous meaning because decreased levels of C3 and C4 reflect inflammation and because they are also prognostic biomarkers for abortion. Furthermore, increased C3 and C4 levels indicate hepatic protein synthesis in hepatocytes. Anti-phospholipid (APL) antibodies are present in 25% to 50% of lupus patients, and can lead to thrombotic and obstetric complications in some pregnancies and increase the risk of abortion, especially in a pregnant woman in the active phase of lupus. Several studies have associated APL with HELLP syndrome. However, other pregnancy complications have not been associated with APL. Autoantibodies against the major vault protein and anti-double strand DNA antibodies are valuable biomarkers in evaluating lupus activity. The expression pattern of micro-RNAs (miRs) differs in various diseases. Current studies have demonstrated the potential of miRs as diagnostic and prognostic biomarkers in various diseases; for example, the level of miR-126 is higher in lupus.

Conclusion

Mir-223-3p and miR-451 are informative biomarkers in estimating disease activity. TWEAK, BAFF, and APOL1 genes, and their polymorphisms are informative in estimating disease activity, especially renal effects, and in monitoring higher-risk pregnant women. Further studies of these genes and their relevant polymorphisms are needed.

     

Isolation of Adenylate Cyclase Gene-Specific Sequences from Ophiostoma novo-ulmi, Candida albicans, and Agaricus bisporus by PCR

Degenerate primers corresponding to consensus sequences in the catalytic domains of known fungal adenylate cyclases were used to isolate gene-specific homologs from the Dutch elm disease pathogen Ophiostoma novo-ulmi, the dimorphic human pathogen Candida albicans, and the commercial mushroom Agaricus bisporus. All three fungi gave the expected PCR product of about 390 bp. Computer searches of the databases revealed that the products generated from O. novo-ulmi and C. albicans were highly similar to the adenylate cyclase gene of Magnaporthe grisea, the rice blast fungus (91% and 79%, respectively). The PCR product from the homobasidiomycete A. bisporus, on the other hand, showed 78% similarity to the uac1 gene of the heterobasidiomycete smut fungus, Ustilago maydis. Southern hybridization indicated that all three fungi contain a single adenylate cyclase gene. Our data suggest that PCR will be highly successful for the isolation of adenylate cyclase sequences from other fungi. Received: 13 April 1998 / Accepted: 27 May 1998     

Competitive interaction between Cotesia kazak and Hyposoter didymator, exotic parasitoids of Heliothis armigera

Competitive interaction between two introduced parasitoids of Heliothis armigera (Hb.) i.e. the braconid Cotesia kazak Telenga and the ichneumonid Hyposoter didymator (Thunb.) was studied in the laboratory. C. kazak and H. didymator individually parasitized about 55% H. armigera larvae, the combination of the two parasitoids gave more than 75% parasitism. C. kazak was found to be more effective, when the host was exposed to both parasitoids at the same time, reduced H. didymator population by 69% to 24 h. It is, therefore, better to release these two parasitoids individually in different locations to avoid any possible competitive interaction between the two. No difference in developmental time was observed irrespective of parasitoid sequence.

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Résumé Les interactions liées à la compétition entre deux parasitoïdes introduits d'H. armigera, le braconide C. kazak et l'ichneumonide H. didymator ont été examinées au laboratoire pour évaluer leur efficacité dans la nature. Séparément, C. kazak et H. didymator ont parasité respectivement 55% et 58% des chenilles d'H. armigera, et en combinaison, par multiparasitisme, 75%. Dans toutes les combinaisons de superparasitisme, après 24 h d'exposition, C. kazak a réduit de plus de 69% l'action de H. didymator. En plus de la mortalité des hôtes liées au succès du parasitisme, une mortalité larvaire élevée (23%) a été observée dans les cas de multiparasitisme, contre 10% avec parasitisme par une espèce. Cependant, quelle que soit la combinaison, la durée de développement de chacune des espèces n'a pas été modifiée. Puisque H. didymator s'est révélé intrinsèquement inférieur, il est suggéré de la lâcher le premier. C. kazak est un parasitoïde valable pour des libérations, là où déplacement compétitif de l'autre parasite n'est pas réalisé ou délibérément nécessaire.