Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase

Saccharomyces cerevisiae utilizes several regulatory mechanisms to maintain tight control over the intracellular level of farnesyl diphosphate (FPP), the central precursor to nearly all yeast isoprenoid products. High-level production of non-native isoprenoid products requires that FPP flux be diverted from production of sterols to the heterologous metabolic reactions. To do so, expression of the gene encoding squalene synthase (ERG9), the first committed step in sterol biosynthesis, was down-regulated by replacing its native promoter with the methionine-repressible MET3 promoter. The intracellular levels of FPP were then assayed by expressing the gene encoding amorphadiene synthase (ADS) and converting the FPP to amorphadiene. Under certain culture conditions amorphadiene production increased fivefold upon ERG9 repression. With increasing flux to amorphadiene, squalene and ergosterol production each decreased. The levels of these three metabolites were dependent not only upon the level of ERG9 repression, but also the timing of its repression relative to the induction of ADS and genes responsible for enhancing flux to FPP.     

Fas-induced changes in cdc2 and cdk2 kinase activity are not sufficient for triggering apoptosis in HUT-78 cells

Recent evidence suggested a role for the cell cycle dependent kinases cdc2 and cdk2 in apoptosis. An important mechanism by which many cell types could undergo apoptosis is through the activation of the Fas molecule on the cell membrane. To investigate whether Fas-induced cell death activated cdc2 and cdk2 kinases inappropriately, the human T lymphoma cells HUT-78, which express a high copy number of Fas, and two other previously characterized subclones of the same cell line which express mutant, cell death-deficient dominant-negative forms of Fas, were Fas-challenged and the changes in cdc2 and cdk2 kinase activity monitored. In both wild-type and Fas-mutated HUT-78 cells, apoptosis was associated simultaneously with decreased cdc2 and increased cdk2 activity. This association suggested that changes in cdc2 and cdk2 kinase activity are secondary events in cell death mediated by Fas. J. Cell. Biochem. 64:579–585. © 1997 Wiley-Liss, Inc.     

Identification of the Nucleotidase Responsible for the AMP Hydrolysing Hyperactivity Associated with Neurological and Developmental Disorders

Nucleoside monophosphate phosphohydrolases comprise a family of enzymes dephosphorylating nucleotides both in intracellular and extracellular compartments. Members of this family exhibit different sequence, location, substrate specificity and regulation. Besides the ectosolic 5′-nucleotidase, several cytosolic and one mitochondrial enzymes have been described. Nevertheless, researchers refer any AMP-dephosphorylating activity to as 5′-nucleotidase, lacking a more accurate identification. Increase of AMP hydrolysing activity has been associated with neurological and developmental disorders. The identification of the specific enzyme involved in these pathologies would be fundamental for the comprehension of the linkage between the enzyme activity alteration and brain functions. We demonstrate that the described neurological symptoms are associated with increased ectosolic 5′-nucleotidase activity on the basis of radiochemical assays and immunoblotting analysis. Furthermore, present data evidence that the assay conditions normally applied for the determination of cytosolic 5′-nucleotidases activity in crude extracts are affected by the presence of solubilised ectosolic nucleotidase.     

Novel integrated microdialysis-amperometric system for in vitro detection of dopamine secreted from PC12 cells: design, construction, and validation

A novel dual channel in vitro apparatus, derived from a previously described design, has been coupled with dopamine (DA) microsensors for the flow-through detection of DA secreted from PC12 cells. The device, including two independent microdialysis capillaries, was loaded with a solution containing PC12 cells while a constant phosphate-buffered saline (PBS) medium perfusion was carried out using a dual channel miniaturized peristaltic pump. One capillary was perfused with normal PBS, whereas extracellular calcium was removed from extracellular fluid of the second capillary. After a first period of stabilization and DA baseline recording, KCl (75 mM) was added to the perfusion fluid of both capillaries. In this manner, a simultaneous “treatment–control” experimental design was performed to detect K⁺-evoked calcium-dependent DA secretion. For this purpose, self-referencing DA microsensors were developed, and procedures for making, testing, and calibrating them are described in detail. The electronic circuitry was derived from previously published schematics and optimized for dual sensor constant potential amperometry applications. The microdialysis system was tested and validated in vitro under different experimental conditions, and DA secretion was confirmed by high-performance liquid chromatography with electrochemical detection (HPLC–EC). PC12 cell viability was quantified before and after each experiment. The proposed apparatus serves as a reliable model for studying the effects of different drugs on DA secretion through the direct comparison of extracellular DA increase in treatment–control experiments performed on the same initial PC12 cell population.     

Spatial and temporal variations in floral resource availability affect bumblebee communities in heathlands

Modifications of landscape structure and composition can decrease the availability of floral resources, resulting in the decline of many pollinator species, including bumblebees. These declines may have significant ecological consequences, because bumblebees pollinate a large range of plant species. Our study was carried out in heathlands, open semi-natural habitats that have decreased considerably due to human activities. We analysed how floral resources affect bumblebee communities throughout the colony lifetime at three scales: plot scale, heathland patch scale, and landscape scale. Floral density at the plot scale and spruce plantations at the landscape scale influenced bumblebee communities. The abundance of bumblebees on ericaceous species was higher when the landscape included a substantial proportion of unsuitable foraging habitat (i.e., spruce plantations). Both life history traits and colony life cycle stage influenced bumblebee responses to the availability of floral resources. Bumblebees were more affected by floral resources during the colony development phase than during the nest-foundation or mating phases. Moreover, bumblebees of species that form large colonies needed larger quantities of favourable foraging habitat, compared with small-colony bees, and their proportion decreased in habitats dominated by spruce plantations. In conclusion, the conservation of plant–bumblebee interactions will require management at a larger spatial scale than the restricted protected habitats. Moreover, at the landscape scale, both quantity of favourable foraging patches and their ecological continuity are important to conserve both small- and large- colony species.     

Non-dermatophytic moulds: onychomycosis in four patients infected with the human immunodeficiency virus

Patients infected with human immunodeficiency virus (HIV) are a risk group for onychomycosis, fungal infections caused mainly by dermatophytes and yeast. However, non-dermatohytic moulds are becoming common agents for nail infections in this population of patients. We report four cases of onychomycosis caused by non-dermatophytic moulds (Aspergillus niger, Scytalidium hyalinum, Scytalidium dimidiatum and Fusarium solani) in patients infected with HIV from Recife, Pernambuco, Brazil. Onychomicosis by non-dermatophytic species in HIV-positive patients requires special attention in the clinical and the laboratory. A proper diagnosis is necessary to establish the specific and adequate treatment, preventing fungal invasion.     

Thromboembolism after COVID-19 vaccine in patients with preexisting thrombocytopenia

While vaccination is the single most effective intervention to drastically reduce severe disease and death following SARS-CoV-2 infection, as shown in UK and Israel, some serious concerns have been raised for an unusual adverse drug reaction (ADR), including vaccine-induced immune thrombotic thrombocytopenia (VITT) with concurrent low platelets as well as capillary leak syndrome. In fact, the overall safety of the vaccine is highlighted by the low frequency of ADR considering that in UK, by the early June, 40 million first doses and 29 million second doses have been injected; nonetheless, 390 thrombotic events, including 71 fatal events have been reported. Interestingly, the cases reported low platelet counts with the presence of anti-platelet factor-4 (PF4) antibodies, indicating an abnormal clotting reaction. Here, out of three referred cases, we report a post-vaccine clinical case of fatal thrombosis with postmortem examination and whole exome sequencing (WES) analysis, whose pathogenesis appeared associated to a preexisting condition of thrombocytopenia due to myelodysplasia.Subject terms: Diseases, Medical research     

Human cytomegalovirus glycoprotein B genotyping from bronchoalveolar lavage specimens

The genes encoding glycoprotein complexes of human cytomegalovirus are often polymorphic; in particular, glycoprotein B (gB), which is essential for both in vivo and in vitro replication, is encoded by the highly polymorphic gene UL55. In this study, the distribution of gB genotypes was investigated in 44 bronchoalveolar lavage specimens from adult patients positive for human cytomegalovirus DNA by a multiplex nested fast PCR able to amplify 5 gB genotypes (gB1-gB5). The distribution of gB genotypes was as follows: 12 (27.3%) gB1, 11 (25%) gB2, 9 (20.4%) gB3, 4 (9.1%) gB4, 0 gB5, and 8 (18.2%) mixed genotypes. No difference in prevalence was found in relation to clinical features, including immunological status, non-transplant or transplant condition, and type of transplanted organ, or in follow-up specimens; while gB4 and gB3 were shown to be significantly more prevalent in patients with respiratory insufficiency, and gB4 and gB2 in those with pneumonia. The prevalence of gB genotypes in the lower respiratory tract was similar to that previously reported using other specimen types and patients, with gB1 found to be the most prevalent. The association of gB genotypes with specific clinical features should be further investigated.