PKH26 Staining Defines Distinct Subsets of Normal Human Colon Epithelial Cells at Different Maturation Stages

BACKGROUND AND AIM: Colon crypts are characterized by a hierarchy of cells distributed along the crypt axis. Aim of this paper was to develop an in vitro system for separation of epithelial cell subsets in different maturation stages from normal human colon. METHODOLOGY AND MAJOR FINDINGS: Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKH(pos) population survived in culture and formed spheroids; this population included subsets with slow (PKH(high)) and rapid (PKH(low)) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr-5 in PKH(high) cells; by cytofluorimetric analysis, Msi-1(+)/Lgr5(+) cells were only found within PKH(high) cells, whereas Msi-1(+)/Lgr5(-) cells were also observed in the PKH(low) population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1(+)/Lgr5(+) cells. While CK20 expression was mainly found in PKH(low) and PKH(neg) cells, a small PKH(high) subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define several subsets at different maturation stages: PKH(high)/Lgr5(+)/Msi-1(+)/CK20(-), PKH(high)/Lgr5(-)/Msi-1(+)/CK20(+), PKH(low)/Lgr5(-)/Msi-1(+)/Ck20(-), and PKH(low)/Lgr5(-)/Msi-1(-)/CK20(+) cells. CONCLUSIONS: Our data show the possibility of deriving in vitro, without any selection strategy, several distinct cell subsets of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in a discrete crypt location.     

Metabolic response of yellow mealworm larvae to two alternative rearing substrates

Introduction

Bitter melon (Momordica charantia, Cucurbitaceae) is a popular edible medicinal plant, which has been used as a botanical dietary supplement for the treatment of diabetes and obesity in Chinese folk medicine. Previously, our team has proved that cucurbitanes triterpenoid were involved in bitter melon’s anti-diabetic effects as well as on increasing energy expenditure. The triterpenoids composition can however be influenced by changes of varieties or habitats.

Objectives

To clarify the significance of bioactive metabolites diversity among different bitter melons and to provide a guideline for selection of bitter melon varieties, an exploratory study was carried out using a UHPLC-HRMS based metabolomic study to identify chemotypes.

Methods

Metabolites of 55 seed samples of bitter melon collected in different parts of China were profiled by UHPLC-HRMS. The profiling data were analysed with multivariate (MVA) statistical methods. Principle component analysis (PCA) and hierarchical cluster analysis (HCA) were applied for sample differentiation. Marker compounds were identified by comparing spectroscopic data with isolated compounds, and additional triterpenes were putatively identified by propagating annotations through a molecular network (MN) generated from UHPLC-HRMS & MS/MS metabolite profiling.

Results

PCA and HCA provided a good discrimination between bitter melon samples from various origins in China. This study revealed for the first time the existence of two chemotypes of bitter melon. Marker compounds of those two chemotypes were identified at different MSI levels. The combined results of MN and MVA demonstrated that the two chemotypes mainly differ in their richness in cucurbitane versus oleanane triterpenoid glycosides (CTGs vs. OTGs).

Conclusion

Our finding revealed a clear chemotype distribution of bioactive components across bitter melon varieties. While bioactivities of individual CTGs and OTGs still need to be investigated in more depth, our results could help in future the selection of bitter melon varieties with optimised metabolites profile for an improved management of diabetes with this popular edible Chinese folk medicine.

     

Unexpected similarity of intestinal sugar absorption by SGLT1 and apical GLUT2 in an insect (Aphidius ervi, Hymenoptera) and mammals

Sugars are critical substrates for insect metabolism, but little is known about the transporters and epithelial routes that ensure their constant supply from dietary resources. We have characterized glucose and fructose uptakes across the apical and basolateral membranes of the isolated larval midgut of the aphid parasitoid Aphidius ervi. The uptake of radiolabeled glucose at the basal side of the epithelium was almost suppressed by 200 microM cytochalasin B, uninhibited by phlorizin, and showed the following decreasing rank of specificity for the tested substrates: glucose > glucosamine > fructose, with no recognition of galactose. These functional properties well agree with the expression of GLUT2-like transporters in this membrane. When the apical surface of the epithelium was also exposed to the labeled medium, a cation-dependent glucose uptake, inhibited by 10 microM phlorizin and by an excess of galactose, was detected suggesting the presence in the apical membrane of a cation-dependent cotransporter. Radiolabeled fructose uptakes were only partially inhibited by cytochalasin B. SGLT1-like and GLUT5-like transporters were detected in the apical membranes of the epithelial cell by immunocytochemical experiments. These results, along with the presence of GLUT2-like transporters both in the apical and basolateral cell membranes of the midgut, as we recently demonstrated, allow us to conclude that the model for sugar transepithelial transport in A. ervi midgut appears to be unexpectedly similar to that recently proposed for sugar intestinal absorption in mammals.     

Identification of H ferritin-dependent and independent genes in K562 differentiating cells by targeted gene silencing and expression profiling

Ferritin is best known as the key molecule in intracellular iron storage, and is involved in several metabolic processes such as cell proliferation, differentiation and neoplastic transformation. We have recently demonstrated that the shRNA silencing of the ferritin heavy subunit (FHC) in a melanoma cell line is accompanied by a consistent modification of gene expression pattern leading to a reduced potential in terms of proliferation, invasiveness, and adhesion ability of the silenced cells.     

A Real Time PCR Platform for the Simultaneous Quantification of Total and Extrachromosomal HIV DNA Forms in Blood of HIV-1 Infected Patients

Background

The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed.

Methodology/Findings

Primers used for total HIV-1 DNA quantification spanning a highly conserved region were selected and found to detect all HIV-1 clades of group M and the unintegrated forms of the same. A total of 195 samples from HIV-1 patients in a wide range of clinical conditions were analyzed with a 100% success rate, even in patients with suppressed plasma viremia, regardless of CD4+ or therapy. No significant correlation was observed between the two current prognostic markers, CD4+ and plasma viremia, while a moderate or high inverse correlation was found between CD4+ and total HIV DNA, with strong values for unintegrated HIV DNA.

Conclusions/Significance

Taken together, the results support the use of HIV DNA as another tool, in addition to traditional assays, which can be used to estimate the state of viral infection, the risk of disease progression and to monitor the effects of ART. The TotUFsys platform allowed us to obtain a final result, expressed as the total and unintegrated HIV DNA copy number per microgram of DNA or 10⁴ CD4+, for 12 patients within two working days.     

Phenotypic diversity,population structure and stress protein-based capacitoring in populations of Xeropicta derbentina,a heat-tolerant land snail species

The shell colour of many pulmonate land snail species is highly diverse. Besides a genetic basis, environmentally triggered epigenetic mechanisms including stress proteins as evolutionary capacitors are thought to influence such phenotypic diversity. In this study, we investigated the relationship of stress protein (Hsp70) levels with temperature stress tolerance, population structure and phenotypic diversity within and among different populations of a xerophilic Mediterranean snail species (Xeropicta derbentina). Hsp70 levels varied considerably among populations, and were significantly associated with shell colour diversity: individuals in populations exhibiting low diversity expressed higher Hsp70 levels both constitutively and under heat stress than those of phenotypically diverse populations. In contrast, population structure (cytochrome c oxidase subunit I gene) did not correlate with phenotypic diversity. However, genetic parameters (both within and among population differences) were able to explain variation in Hsp70 induction at elevated but non-pathologic temperatures. Our observation that (1) population structure had a high explanatory potential for Hsp70 induction and that (2) Hsp70 levels, in turn, correlated with phenotypic diversity while (3) population structure and phenotypic diversity failed to correlate provides empirical evidence for Hsp70 to act as a mediator between genotypic variation and phenotype and thus for chaperone-driven evolutionary capacitance in natural populations.     

Towards cell‐free isobutanol production: Development of a novel immobilized enzyme system

Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell‐free immobilized enzyme system. Two immobilized enzymes (keto‐acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:66–73, 2016     

The possible role of chromogranin A as a prognostic factor in organ-confined prostate cancer

The clinical significance of neuroendocrine differentiation in patients who have undergone surgery for localized prostate cancer is still unclear. The aims of this study were to assess the relationship between serum neuroendocrine markers and well-known prognostic factors in prostate cancer (pathological staging, definitive Gleason score and serum PSA) and to search for correlations between serum chromogranin A (CgA) levels and pathological findings. Forty-one consecutive patients who had undergone radical retropubic prostatectomy for clinically localized prostate cancer were evaluated. Serum PSA, CgA and neuron-specific enolase were measured immediately before surgery. Twenty-six surgical specimens were phenotypically and immunohistochemically evaluated using an antibody against CgA. Significant correlations were found between serum CgA, pathological staging and Gleason score (p=0.049 and p=0.038, respectively). Serum CgA did not correlate with PSA, patient age, or immunohistochemical findings. There was a significant correlation between positive immunohistochemical CgA staining and Gleason score (p=0.014). An increase in serum CgA levels, independent of PSA values, might be the expression of pathologically more advanced tumor stage and higher Gleason score; this could help to identify a high-risk patient group eligible for adjuvant therapy.     

Cadmium induces changes in corticotropic and prolactin cells in the Podarcis sicula lizard pituitary gland

We analyzed the effect of cadmium on corticotropic (ACTH) and prolactin (PRL) cells in the pituitary gland of the Podarcis sicula (P. sicula) lizard under chronic exposure to this metal. Adult lizards were given CdCl₂ in drinking water at the dose of 10 µg/10 g body mass for 120 days. Light microscopy was performed after histological and immunohistochemical staining, and the effects were followed at regular time intervals up to 120 days post-treatment. We detected substantial variations in the general morphology of the pituitary: unlike the control lizards in which the gland appeared compact, the treated lizards showed a glandular tissue with dilated spaces that were more extensive at 90 and 120 days. PRL and ACTH cells showed an increase in occurrence and immunostaining intensity in treated lizards in comparison with the same cells of control animals. This cellular increase peaked for PRL at 30 days in the rostral, medial and also caudal pars distalis of the gland. ACTH cells appeared to increase markedly after 60 days of treatment in both the pars distalis and the pars intermedia. Again, at 60 days small, isolated ACTH cells were also found in the caudal pars distalis in which these cells were generally absent. However, at 120 days both these cellular types showed an occurrence, distribution and morphology similar to those observed in the control lizards. In lizards, protracted oral exposure to cadmium evidently involves an alteration of the normal morphology of the gland and an inhibitory effect of ACTH and PRL cells, since they increase in occurrence and immunostaining. Yet in time the inhibitory effect of cadmium on ACTH and PRL cells falls back and their occurrence appears similar to that of the control lizard.Key words: cadmium, corticotropic and prolactin cells, lizard.