Protochlorophyllide-independent import of two NADPH:Pchlide oxidoreductase proteins (PORA and PORB) from barley into isolated plastids

The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), NADPH:Pchlide oxidoreductase (POR; EC 1.6.99.1), is a nuclear-encoded protein that is post-translationally imported to the plastid. In barley and Arabidopsis thaliana , the reduction of Pchlide is controlled by two different PORs, PORA and PORB. To characterise the possible Pchlide dependency for the import reaction, radiolabelled precursor proteins of barley PORA and PORB (pPORA and pPORB, respectively) were used for in vitro assays with isolated plastids of barley and pea with different contents of Pchlide. To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley plants were grown in darkness or in greenhouse conditions for 6 days. Alternatively, greenhouse-grown pea plants were incubated for 4 days in darkness before plastid isolation, or chloroplasts isolated from greenhouse-grown plants were incubated with Δ -aminolevulinic acid (ALA), an early precursor in the Chl biosynthesis resulting in elevated Pchlide contents in the plastids. Both barley pPORA and pPORB were effectively imported into barley and pea chloroplasts isolated from the differentially treated plants, including those isolated from greenhouse-grown plants. The absence or presence of Pchlide did not significantly affect the import capacity of barley pPORA or pPORB. Assays performed on stroma-enriched fractions from chloroplasts and etioplasts of barley indicated that no post-import degradation of the proteins occurred in the stroma, irrespective of whether the incubation was performed in darkness or in light.     

Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

IntroductionThe clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing.Material and methodsIndividual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes.ResultsThe prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients.Conclusion and recommendationOur findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.     

Biased Signaling at Chemokine Receptors

The ability of G protein-coupled receptors (GPCRs) to activate selective signaling pathways according to the conformation stabilized by bound ligands (signaling bias) is a challenging concept in the GPCR field. Signaling bias has been documented for several GPCRs, including chemokine receptors. However, most of these studies examined the global signaling bias between G protein- and arrestin-dependent pathways, leaving unaddressed the potential bias between particular G protein subtypes. Here, we investigated the coupling selectivity of chemokine receptors CCR2, CCR5, and CCR7 in response to various ligands with G protein subtypes by using bioluminescence resonance energy transfer biosensors monitoring directly the activation of G proteins. We also compared data obtained with the G protein biosensors with those obtained with other functional readouts, such as β-arrestin-2 recruitment, cAMP accumulation, and calcium mobilization assays. We showed that the binding of chemokines to CCR2, CCR5, and CCR7 activated the three Gα_i subtypes (Gα_i1, Gα_i2, and Gα_i3) and the two Gα_o isoforms (Gα_oa and Gα_ob) with potencies that generally correlate to their binding affinities. In addition, we showed that the binding of chemokines to CCR5 and CCR2 also activated Gα₁₂, but not Gα₁₃. For each receptor, we showed that the relative potency of various agonist chemokines was not identical in all assays, supporting the notion that signaling bias exists at chemokine receptors.     

Facile removal of 4‐methoxybenzyl protecting group from selenocysteine

Historically, methods to remove the 4‐methoxybenzyl (Mob)–protecting group from selenocysteine (Sec) in peptides have used harsh and toxic reagents. The use of 2,2′‐dithiobis‐5‐nitropyridine (DTNP) is an improvement over these methods; however, many wash steps are required to remove the by‐product contaminant 5‐nitro‐2‐thiopyridine. Even with many washes, excess DTNP adheres to the peptide. The final product needs excess purification to remove these contaminants. It was recently discovered by our group that hindered hydrosilanes could be used to reduce Cys(Mob). We sought to apply a similar methodology to reduce Sec(Mob), which we expected to be even more labile. Here, we present a gentle and facile method for deprotection of Sec(Mob) using triethylsilane (TES), phenol, and a variety of other scavengers often used in deprotection cocktails. The different cocktails were all incubated at 40 °C for 4 hours. The combination of TFA/TES/thioanisole (96:2:2) appeared to be the most efficient of the cocktails tested, providing complete deprotection and yielded peptide that was mainly in the diselenide form. This cocktail also showed no evidence of side reactions or significant contaminants in the high‐performance liquid chromatography (HPLC) and mass spectral (MS) analyses. We envision that our new method will allow for a simple and gentle “one‐pot” deprotection of Sec(Mob) following solid‐phase peptide synthesis and will minimize the need for extensive purification steps.     

Specific Chaperones and Regulatory Domains in Control of Amyloid Formation

Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology.     

Substantial advantage of a combined Bayesian and genotyping approach in testosterone doping tests

Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. A deletion polymorphism in the gene coding for UGT2B17 is strongly associated with reduced testosterone glucuronide (TG) levels in urine. Many of the individuals devoid of the gene would not reach a T/E ratio of 4.0 after testosterone intake.Future test programs will most likely shift from population based- to individual-based T/E cut-off ratios using Bayesian inference. A longitudinal analysis is dependent on an individual's true negative baseline T/E ratio.The aim was to investigate whether it is possible to increase the sensitivity and specificity of the T/E test by addition of UGT2B17 genotype information in a Bayesian framework.A single intramuscular dose of 500 mg testosterone enanthate was given to 55 healthy male volunteers with either two, one or no allele (ins/ins, ins/del or del/del) of the UGT2B17 gene.Urinary excretion of TG and the T/E ratio was measured during 15 days.The Bayesian analysis was conducted to calculate the individual T/E cut-off ratio.When adding the genotype information, the program returned lower individual cut-off ratios in all del/del subjects increasing the sensitivity of the test considerably.It will be difficult, if not impossible, to discriminate between a true negative baseline T/E value and a false negative one without knowledge of the UGT2B17 genotype.UGT2B17 genotype information is crucial, both to decide which initial cut-off ratio to use for an individual, and for increasing the sensitivity of the Bayesian analysis.     

Blowing in the wind? Identity, materiality, and the destinations of human ashes

This article asks whether the recent UK-based practice of removing ashes from crematoria has led to entirely new, innovative rituals of disposal, or whether contemporary practice is an appropriation of late nineteenth-century Romantic values and beliefs. Drawing on findings from a major empirical study among both professionals and lay people involved in the removal of ashes, it explores the potentiality of ash remains as a mobile material residue of the corpse, and considers whether they enable disposal strategies which no longer reflect concerns with space and place – particularly those associated with traditional burial grounds.     

Neuronal Cx3cr1 Deficiency Protects against Amyloid β-Induced Neurotoxicity

Cx3cr1, the receptor for the chemokine Cx3cl1 (fractalkine), has been implicated in the progression and severity of Alzheimer’s disease-like pathology in mice, but the underlying mechanisms remain unclear. A complicating factor is that Cx3cr1 has been demonstrated in both neurons and microglia. Here, we have dissected the differences between neuronal and microglial Cx3cr1, specifically by comparing direct amyloid-β-induced toxicity in cultured, mature, microglia-depleted hippocampal neurons from wild-type and Cx3cr1^-/- mice. Wild-type neurons expressed both Cx3cl1 and Cx3cr1 and released Cx3cl1 in response to amyloid-β. Knockout of neuronal Cx3cr1 abated amyloid-β-induced lactate dehydrogenase release. Furthermore, amyloid-β differentially induced depression of pre- and postsynaptic components of miniature excitatory postsynaptic currents, in a peptide conformation-dependent manner. Knockout of neuronal Cx3cr1 abated effects of both amyloid-β conformational states, which were differentiable by aggregation kinetics and peptide morphology. We obtained similar results after both acute and chronic treatment of cultured neurons with the Cx3cr1 antagonist F1. Thus, neuronal Cx3cr1 may impact Alzheimer’s disease-like pathology by modulating conformational state-dependent amyloid-β-induced synaptotoxicity.     

Dietary Mechanism behind the Costs Associated with Resistance to Bacillus thuringiensis in the Cabbage Looper,Trichoplusia ni

Beneficial alleles that spread rapidly as an adaptation to a new environment are often associated with costs that reduce the fitness of the population in the original environment. Several species of insect pests have evolved resistance to Bacillus thuringiensis (Bt) toxins in the field, jeopardizing its future use. This has most commonly occurred through the alteration of insect midgut binding sites specific for Bt toxins. While fitness costs related to Bt resistance alleles have often been recorded, the mechanisms behind them have remained obscure. We asked whether evolved resistance to Bt alters dietary nutrient intake, and if reduced efficiency of converting ingested nutrients to body growth are associated with fitness costs and variation in susceptibility to Bt. We fed the cabbage looper Trichoplusia ni artificial diets differing in levels of dietary imbalance in two major macronutrients, protein and digestible carbohydrate. By comparing a Bt-resistant T. ni strain with a susceptible strain we found that the mechanism behind reduced pupal weights and growth rates associated with Bt-resistance in T. ni was reduced consumption rather than impaired conversion of ingested nutrients to growth. In fact, Bt-resistant T. ni showed more efficient conversion of nutrients than the susceptible strain under certain dietary conditions. Although increasing levels of dietary protein prior to Bt challenge had a positive effect on larval survival, the LC₅₀ of the resistant strain decreased when fed high levels of excess protein, whereas the LC₅₀ of the susceptible strain continued to rise. Our study demonstrates that examining the nutritional basis of fitness costs may help elucidate the mechanisms underpinning them.