Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells

It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks.     

Correction to: Who is the ocean? Preface to the future seas 2030 special issue

Reviews in Fish Biology and Fisheries - A correction to this paper has been published: https://doi.org/10.1007/s11160-021-09657-9     

Recurrent hybridisation events between Primula vulgaris,P. veris and P. elatior (Primulaceae,Ericales) challenge the species boundaries: using molecular markers to re‐evaluate morphological identifications

Three Primula species, Primula vulgaris, P. veris and P. elatior, have been objects of fascination for gardeners and botanists over several centuries. The species are able to hybridise, and where they co‐occur, hybrids are commonly found. In Denmark, Møns Klint on the island of Møn and Købelev Skov on Lolland are examples of localities where all three species occur and where the hybrids P. × digenea, the hybrid between P. vulgaris and P. elatior, and P. × polyantha, the hybrid between P. veris and P. vulgaris, can also be found. To investigate relationships between the species and their hybrids, we sampled 168 specimens from 10 geographical locations and subjected them to genetic analysis. The samples were also identified based on morphological traits: primarily inflorescense structure, the size, shape, colour and markings of corolla and leaf basis, leaf blade texture and hairiness. After identifying species‐specific SNPs in the Internal Transcribed Spacer sequence, these were used to resolve species and hybrid boundaries and status through a cleaved amplified polymorphic sequence assay. Polymorphisms in the chloroplast trnL sequence were used as a high‐throughput marker and used to determine the maternal parent of hybrids. Ten simple sequence repeat markers were applied to obtain further insight into the genetic makeup of the accessions using structure and Introgress, providing information of genetic variability within and between populations. Our results indicated that backcrossing of P. × digenea hybrids with parental species has occurred, and that many of the P. × digenea sampled were later‐generation hybrids rather than F₁s. Analyses of P. × polyantha specimens show mostly the expected pattern for primary hybrids but indications of P. veris ancestry of a P. vulgaris plant was discovered. Our results further indicate that some of the specimens initially identified as P. elatior include P. vulgaris among their progenitors and thus challenge currently accepted species boundaries.     

Microbial growth and accumulation in industrial metal-working fluids.

The dynamics of microbial growth in metal-working fluids (MWF) and the effect of the addition of biocides were studied in large fluid systems, in this case, one central tank which holds 150 m3. In this system, populations of Pseudomonas pseudoalcaligenes (greater than 10(8) CFU/ml) were sustained for a year, although large quantities of biocides were added. Quantitation of 3-OH lauric acid, a marker for many Pseudomonas spp., by gas chromatography indicated that the bacterial biomass exceeded the viable counts by approximately 15 times. Fungi were grown on several occasions, the dominating genera being Fusarium and Candida. Soon after the old MWF was removed and the tank was provided with fresh MWF, which consisted of an emulsion of mineral oil in water, there was a massive growth of P. pseudoalcaligenes that reached levels of greater than 10(8) bacteria per ml. Initially, only low concentrations of other species were found for some weeks. After this period, different enterobacteria and other gram-negative rods often appeared at high concentrations (10(7) and 10(8) bacteria per ml, respectively). Bacteria identified as P. pseudoalcaligenes showed great variation with respect to colony morphology and a certain heterogeneity with respect to biochemical characteristics. Certain bacterial species grew as microcolonies on metal strips immersed in the circulating MWF, but P. pseudoalcaligenes was not recovered from this habitat. The total bacterial count in the air surrounding the machines in the metal-working shop showed an inverse relation to increasing distance from the machine. The concentration of bacteria in the air varied because of the number of machines in use, temperature, and humidity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaccination with p53-peptide–pulsed dendritic cells,of patients with advanced breast cancer: report from a phase I study

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2⁺, p53⁺ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2⁺ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.     

Rapid Implementation of an Integrated Large-Scale HIV Counseling and Testing,Malaria, and Diarrhea Prevention Campaign in Rural Kenya

Background

Integrated disease prevention in low resource settings can increase coverage, equity and efficiency in controlling high burden infectious diseases. A public-private partnership with the Ministry of Health, CDC, Vestergaard Frandsen and CHF International implemented a one-week integrated multi-disease prevention campaign.

Method

Residents of Lurambi, Western Kenya were eligible for participation. The aim was to offer services to at least 80% of those aged 15–49. 31 temporary sites in strategically dispersed locations offered: HIV counseling and testing, 60 male condoms, an insecticide-treated bednet, a household water filter for women or an individual filter for men, and for those testing positive, a 3-month supply of cotrimoxazole and referral for follow-up care and treatment.

Findings

Over 7 days, 47,311 people attended the campaign with a 96% uptake of the multi-disease preventive package. Of these, 99.7% were tested for HIV (87% in the target 15–49 age group); 80% had previously never tested. 4% of those tested were positive, 61% were women (5% of women and 3% of men), 6% had median CD4 counts of 541 cell/µL (IQR; 356, 754). 386 certified counselors attended to an average 17 participants per day, consistent with recommended national figures for mass campaigns. Among women, HIV infection varied by age, and was more likely with an ended marriage (e.g. widowed vs. never married, OR.3.91; 95% CI. 2.87–5.34), and lack of occupation. In men, quantitatively stronger relationships were found (e.g. widowed vs. never married, OR.7.0; 95% CI. 3.5–13.9). Always using condoms with a non-steady partner was more common among HIV-infected women participants who knew their status compared to those who did not (OR.5.4 95% CI. 2.3–12.8).

Conclusion

Through integrated campaigns it is feasible to efficiently cover large proportions of eligible adults in rural underserved communities with multiple disease preventive services simultaneously achieving various national and international health development goals.     

Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides.

We have used one and two dimensional 1H NMR spectroscopy to characterize the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4- (3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolin ium tetraiodide (TOTO), to various double stranded DNA oligonucleotides. TOTO binds strongly to all the oligonucleotides used, but usually more than one complex is observed and exchange between different binding sites broadens the lines in the NMR spectra. Complete precipitation occurs when TOTO is bound to small oligonucleotides. Binding to larger oligonucleotides occurs by bis-intercalation. The 1:1 complex of TOTO with the oligonucleotide d(CCGACTGATGC):d (GCATCAGTCGG) gave only one complex that was shown to be a bis-intercalation in the CTGA:TCAG binding site. The binding to this site was also characterized by studying the TOTO complex with the d(CCGCTGAGC):d(GCTCAGCGG) oligonucleotide. NOE connectivities and molecular modelling were used to characterize the complex. The 1:1 complex of TOTO with the oligonucleotide d(CCGCTAGCG):d(CGCTAGCGG) containing a CTAG:CTAG binding site was similarly characterized by NMR. It was concluded that the binding of TOTO to larger oligonucleotides is site selective with CTAG:CTAG as the preferred binding site.