New scoring system to identify RNA G-quadruplex folding

G-quadruplexes (G4s) are non-canonical structures involved in many important cellular processes. To date, the prediction of potential G-quadruplex structures (PG4s) has been based almost exclusively on the sequence of interest agreeing with the algorithm G_x-N-_1–7-G_x-N_1–7-G_x-N_1–7-G_x (where x ≥ 3 and N = A, U, G or C). However, many sequences agreeing with this algorithm do not form G4s and are considered false-positive predictions. Here we show the RNA PG4 candidate in the 3′-untranslated region (UTR) of the TTYH1 gene to be one such false positive. Specifically, G4 folding was observed to be inhibited by the presence of multiple-cytosine tracks, located in the candidate’s genomic context, that adopted a Watson–Crick base-paired structure. Clearly, the neighbouring sequences of a PG4 may influence its folding. The secondary structure of 12 PG4 motifs along with either 15 or 50 nucleotides of their upstream and downstream genomic contexts were evaluated by in-line probing. Data permitted the development of a scoring system for the prediction of PG4s taking into account the effect of the neighbouring sequences. The accuracy of this scoring system was assessed by probing 14 other novel PG4 candidates retrieved in human 5′-UTRs. This new scoring system can be used, in combination with the standard algorithm, to better predict the folding of RNA G4s.     

Kinetic and crystallographic studies on the active site Arg289Lys mutant of flavocytochrome b2 (yeast L-lactate dehydrogenase)

Flavocytochrome b(2) from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction. The crystal structure of the native yeast enzyme has been determined [Xia, Z.-X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 837-863] as well as that of the sulfite adduct of the recombinant enzyme produced in Escherichia coli [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]; several key active site residues were identified. In the sulfite adduct crystal structure, Arg289 adopts two alternative conformations. In one of them, its side chain is stacked against that of Arg376, which interacts with the substrate; in the second orientation, the R289 side chain points toward the active site. This residue has now been mutated to lysine and the mutant enzyme, R289K-b(2), characterized kinetically. Under steady-state conditions, kinetic parameters (including the deuterium kinetic isotope effect) indicate the mutation affects k(cat) by a factor of about 10 and k(cat)/K(M) by up to nearly 10(2). Pre-steady-state kinetic analysis of flavin and heme reduction by lactate demonstrates that the latter is entirely limited by flavin reduction. Inhibition studies on R289K-b(2) with a range of compounds show a general rise in K(i) values relative to that of wild-type enzyme, in line with the elevation of the K(M) for L-lactate in R289K-b(2); they also show a change in the pattern of inhibition by pyruvate and oxalate, as well as a loss of the inhibition by excess substrate. Altogether, the kinetic studies indicate that the mutation has altered the first step of the catalytic cycle, namely, flavin reduction; they suggest that R289 plays a role both in Michaelis complex and transition-state stabilization, as well as in ligand binding to the active site when the flavin is in the semiquinone state. In addition, it appears that the mutation has not affected electron transfer from fully reduced flavin to heme, but may have slowed the second intramolecular ET step, namely, transfer from flavin semiquinone to heme b(2). Finally, the X-ray crystal structure of R289K-b(2), with sulfite bound at the active site, has been determined to 2.75 A resolution. The lysine side chain at position 289 is well-defined and in an orientation that corresponds approximately to one of the alternative conformations observed in the structure of the recombinant enzyme-sulfite complex [Tegoni, M., and Cambillau, C. (1994) Protein Sci. 3, 303-313]. Comparisons between the R289K-b(2) and wild-type structures allow the kinetic results to be interpreted in a structural context.     

Plasmodium falciparum: freeze-fracture of the gametocyte pellicular complex

Freeze-fracturing has been used to study the architecture of the pellicular complex of the gametocytes of Plasmodium falciparum. The gametocyte is surrounded by three membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes is split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on their surfaces. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. In gametocytes, unlike in sporozoites, the intramembranous particles are always distributed randomly and lack any definite pattern or orientations. A unique feature of gametocytes revealed by the freeze-fracturing technique is the presence of several transverse sutures on the middle membrane that encircle the gametocyte and give it a segmented appearance.     

Involvement of Arabidopsis ACYL-COENZYME A DESATURASE-LIKE2 (At2g31360) in the Biosynthesis of the Very-Long-Chain Monounsaturated Fatty Acid Components of Membrane Lipids

The Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A (CoA) desaturase-like (ADS) gene family contains nine genes encoding fatty acid desaturase-like proteins. The biological function of only one member of the family, fatty acid desaturase5 (AtADS3/FAD5, At3g15850), is known, and this gene encodes the plastidic palmitoyl-monogalactosyldiacylglycerol Δ7 desaturase. We cloned seven members of the gene family that are predicted not to have a chloroplast transit peptide and expressed them in the yeast Saccharomyces cerevisiae. All seven have previously undescribed desaturase activity on very-long-chain fatty acid (VLCFA) substrates and exhibit diverse regiospecificity, catalyzing introduction of double bonds relative to the methyl end of the molecule (n-x) at n-6 (AtADS4, At1g06350), n-7 (AtADS1.3, At1g06100 and AtADS4.2, At1g06360), n-9 (AtADS1, At1g06080 and AtADS2, At2g31360) or Δ9 (relative to the carboxyl end of the molecule) positions (AtADS1.2, At1g06090 and AtADS1.4, At1g06120). Through forward and reverse genetics it was shown that AtADS2 is involved in the synthesis of the 24:1(n-9) and 26:1(n-9) components (X:Y, where X is chain length and Y is number of double bonds) of seed lipids, sphingolipids, and the membrane phospholipids phosphatidylserine, and phosphatidylethanolamine. Plants deficient in AtADS2 expression showed no obvious phenotype when grown under normal growing conditions, but showed an almost complete loss of phosphatidylethanolamine(42:4), phosphatidylserine(42:4), dihydroxy-monohexosylceramide(42:2)-2, trihydroxy-monohexosylceramide(42:2)-3, and trihydroxy-glycosylinositolphosphoceramide(42:2)-3, lipid species that contain the VLCFA 24:1(n-9), and trihydroxy-glycosylinositolphosphoceramide(44:2)-3, a lipid containing 26:1(n-9). Acyl-CoA profiling of these plants revealed a major reduction in 24:1-CoA and a small reduction in 26:1-CoA. Overexpression of AtADS2 resulted in a substantial increase in the percentage of glycerolipid and sphingolipids species containing 24:1 and a dramatic increase in the percentage of very-long-chain monounsaturated fatty acids in the acyl-CoA pool. Plants deficient in AtADS1 expression had reduced levels of 26:1(n-9) in seed lipids, but no significant changes in leaf phospholipids or sphingolipids were observed. These findings indicate that the 24-carbon and 26-carbon monounsaturated VLCFAs of Arabidopsis result primarily from VLCFA desaturation, rather than by elongation of long chain monounsaturated fatty acids.The ADS (for acyl-CoA desaturase-like) gene family of Arabidopsis (Arabidopsis thaliana) encodes a group of nine proteins with homology to the Δ9 acyl-lipid desaturases of cyanobacteria, the Δ9 acyl-CoA desaturases of yeast (Saccharomyces cerevisiae) and mammals (Fukuchi-Mizutani et al., 1998; Heilmann et al., 2004b) and the membrane-bound desaturases of insects (Knipple et al., 2002). Eight of these genes are located in three clusters on chromosomes I and III. The remaining gene, designated AtADS2 (At2g31360), is present on chromosome II. With the exception of Arabidopsis fatty acid desaturase5 (AtADS3/FAD5, At3g15850), which encodes the plastidic palmitoyl-monogalactosyldiacylglycerol Δ7 desaturase (Heilmann et al., 2004b), the biological role of these enzymes in Arabidopsis is currently unknown. AtADS3/FAD5 and a second closely linked homolog designated AtADS3.2 (At3g15870), are the only members of the gene family predicted to encode proteins with a plastid transit peptide.The first study, to our knowledge, to report evidence of desaturase activity associated with an Arabidopsis ADS, AtADS1 (At1g06080), described the heterologous expression of the gene in Brassica juncea. Seeds from transformed plants contained decreased levels of saturated fatty acids and a slight increase in oleic acid content (Yao et al., 2003). Although the evidence was indirect, the results suggested that AtADS1 may encode a Δ9 desaturase. More detailed studies involving in vivo expression of AtADS1, AtADS2, and AtADS3 (without the plastid transit peptide) in yeast (Saccharomyces cerevisiae) have shown that all three enzymes can catalyze the Δ9 or Δ7 desaturation of palmitic (16:0) and stearic (18:0) acids (X:Y, where X is chain length and Y is number of double bonds), with regiospecificity being partly influenced by fatty acid substrate (Heilmann et al., 2004a). In this work, the substrate for desaturation was suggested to be a glycerolipid rather than acyl-CoA. The bifunctionality of these enzymes was further demonstrated by expression in Arabidopsis. When AtADS3 was expressed as the full-length form including the plastid transit peptide, or when AtADS1 and AtADS2 were retargeted to the plastid by the addition of a plastid transit peptide, 16:1Δ7 became the predominant monounsaturated 16-carbon (C16) fatty acid. The Arabidopsis plants used in the study were fab1/fae1 (for fatty acid elongase1) double mutant lines lacking the activity of KASII (for 3-ketoacyl-acyl-carrier protein synthase; FAB1, At1g74960) and the FAE1 condensing enzyme (At4g34520), and consequently exhibiting higher than normal levels of 16:0 and low very-long-chain unsaturated fatty acid content in the seed lipids.Homologs of the Arabidopsis ADS enzymes have been identified in other plant species, but their catalytic activity and acyl-substrates are not well characterized. Heterologous expression of a complementary DNA (cDNA) encoding an ADS-like protein from white spruce (Picea glauca) gave evidence of Δ9 activity when expressed in yeast (Marillia et al., 2002). The lipid substrate of this desaturase was not determined and the cDNA appeared to encode an enzyme with a plastid transit peptide. The Δ5 desaturase catalyzing the synthesis of 20:1Δ5 in the seeds of Limnanthes alba is also an ADS homolog (Cahoon et al., 2000). The substrate for this reaction is thought to be the 20:0-CoA thioester (Pollard and Stumpf, 1980; Moreau et al., 1981). Δ5 desaturase activity on fatty acids with chain length longer than 18 carbons (very-long-chain fatty acids [VLCFAs]) has also been demonstrated from two ADS homologs isolated from Anemone leveillei (Sayanova et al., 2007). Indirect evidence suggesting that both enzymes utilized acyl-CoA substrates was presented based on characterization of acyl-CoA pools in developing seeds of transgenic Arabidopsis expressing the A. leveillei desaturases.In addition to functioning in the synthesis of chloroplast lipids (AtADS3/FAD5) and VLCFAs of certain seed oils, ADS proteins have been suggested to play a role in petal senescence in roses (Rosa spp.; Fukuchi-Mizutani et al., 1995) and the expression of Arabidopsis AtADS1 and AtADS2 appears to be regulated in response to changes in temperature (Fukuchi-Mizutani et al., 1998; Byun et al., 2009). A potential role in drought tolerance has also been suggested for a member of the gene family (At1g06100; Allen et al., 2012). This group of plant enzymes therefore appears to contain members showing a diversity of lipid substrate utilization, desaturation regiospecificity and biological function that merits further investigation.Only four members of the Arabidopsis ADS gene family have documented nomenclature. Based on the existing literature we propose a systematic nomenclature of the ADS gene family based on their chromosomal location and grouping (VLCFA substrates. Forward and reverse genetics have revealed a role for AtADS2 in the production of very-long-chain monounsaturated fatty acids (VLCMUFAs) in seed lipids and in membrane phospholipids and sphingolipids.

Table I.

The Arabidopsis ADS gene family

Long-distance travel for birthing among Indigenous and non-Indigenous pregnant people in Canada

BACKGROUND:For Indigenous Peoples in Canada, birthing on or near traditional territories in the presence of family and community is of foundational cultural and social importance. We aimed to evaluate the association between Indigenous identity and distance travelled for birth in Canada.METHODS:We obtained data from the Maternity Experiences Survey, a national population-based sample of new Canadian people aged 15 years or older who gave birth (defined as mothers) and were interviewed in 2006–2007. We compared Indigenous with non-Indigenous Canadian-born mothers and adjusted for geographic and sociodemographic factors and medical complications of pregnancy using multivariable logistic regression. We categorized the primary outcome, distance travelled for birth, as 0 to 49, 50 to 199 or 200 km or more.RESULTS:We included 3100 mothers living in rural or small urban areas, weighted to represent 31 100 (1800 Indigenous and 29 300 non-Indigenous Canadian-born mothers). We found that travelling 200 km or more for birth was more common among Indigenous compared with non-Indigenous mothers (9.8% v. 2.0%, odds ratio [OR] 5.45, 95% confidence interval [CI] 3.52–8.48). In adjusted analyses, the association between Indigenous identity and travelling more than 200 km for birth was even stronger (adjusted OR 16.44, 95% CI 8.07–33.50) in rural regions; however, this was not observed in small urban regions (adjusted OR 1.04, 95% CI 0.37–2.91).INTERPRETATION:Indigenous people in Canada experience striking inequities in access to birth close to home compared with non-Indigenous people, primarily in rural areas and independently of medical complications of pregnancy. This suggests inequities are rooted in the geographic distribution of and proximal access to birthing facilities and providers for Indigenous people.

Access to birth close to home, surrounded by loved ones, is taken for granted by most Canadians. The societal importance of family support during birthing has been highlighted during the severe acute respiratory syndrome and COVID-19 pandemics, despite the known and potentially fatal risks to hospital visitors, because people in labour have been one of the few patient groups exempt from visitor restrictions.^1,2 For residents living in rural areas of Canada, long-distance travel for birth is a reality that is becoming increasingly common in some regions because of closures of obstetrical services in smaller community hospitals.³ This is only partially mitigated by the revitalization of rural midwifery practice.^4,5Emerging evidence shows that the frequency of adverse medical events during labour and delivery for rural populations is similar for births that take place close to home and births for which people travel because of an absence of services close to home.^3,4,6 Less is known about the impacts of travel for birth on breastfeeding rates, maternal mental health and family functioning. Several studies have documented the negative impacts of birthing away from home with respect to maternal satisfaction and birth experience.^7–10 This evidence is particularly compelling for Indigenous populations for whom birthing on or near traditional territories in the presence of family and community is a long-standing practice of foundational cultural and social importance that contributes to well-being, cultural continuity and kinship.^7–12The striking isolation, family disruption and racism experienced by Indigenous people who are forced to travel alone for birth as a result of externally imposed federal “evacuation for birth” policies¹¹ has been met with a series of policy initiatives to support return of birth to rural and remote Indigenous communities. ^13–15 In April 2017, then federal Minister of Health Dr. Jane Philpott, committed to “a path to be able to return the cries of birth” to Indigenous communities and funding to support travel for a companion when Indigenous people living in rural and remote areas needed to travel away from home for birth.¹⁶ Before 2017, Indigenous pregnant people often travelled and birthed away from home alone without family or community support, because escorts were not deemed medically necessary. Although these initiatives have improved access to Indigenous perinatal programming and Indigenous birth attendant support in some local areas, over the past decade there has not been any substantial expansion of Indigenous birthing facilities outside of urban centres in Canada and at least 1 remote Indigenous birthing facility has closed.¹⁷Given this dynamic policy context, the national scope and Indigenous identifiers in the Canadian Maternity Experiences Survey (MES) provides a unique opportunity to quantify how often Indigenous and non-Indigenous people are travelling away from home for birth and to evaluate the association between Indigenous and non-Indigenous identity and distance travelled for birth in Canada.     

Gray matter imaging in multiple sclerosis: what have we learned?

At the early onset of the 20^th century, several studies already reported that the gray matter was implicated in the histopathology of multiple sclerosis (MS). However, as white matter pathology long received predominant attention in this disease, and histological staining techniques for detecting myelin in the gray matter were suboptimal, it was not until the beginning of the 21^st century that the true extent and importance of gray matter pathology in MS was finally recognized. Gray matter damage was shown to be frequent and extensive, and more pronounced in the progressive disease phases. Several studies subsequently demonstrated that the histopathology of gray matter lesions differs from that of white matter lesions. Unfortunately, imaging of pathology in gray matter structures proved to be difficult, especially when using conventional magnetic resonance imaging (MRI) techniques. However, with the recent introduction of several more advanced MRI techniques, the detection of cortical and subcortical damage in MS has considerably improved. This has important consequences for studying the clinical correlates of gray matter damage. In this review, we provide an overview of what has been learned about imaging of gray matter damage in MS, and offer a brief perspective with regards to future developments in this field.     

Overexpression of seleno-glutathione peroxidase by gene transfer enhances the resistance of T47D human breast cells to clastogenic oxidants

The role of seleno-glutathione peroxidase (GSHPx; EC 1.11.1.9) in the cellular defense against oxidative stress was selectively investigated in novel cell models. Expression vectors designed to overexpress human GSHPx efficiently in a broad range of mammalian cells were used to transfect T47D human breast cells which contain very low levels of endogenous GSHPx. Several stable transfectants expressing GSHPx to various extents, up to 10-100 times more than parental cells, were isolated and characterized. Growth inhibition kinetics following transient exposure to increasing concentrations of H2O2, cumene hydroperoxide or menadione (an intracellular source of free radicals and reactive oxygen intermediates) showed that transfectants overexpressing GSHPx were considerably more resistant than control T47D cell derivatives to each of these oxidants. A sensitive DNA end-labeling procedure was used as a novel approach to compare relative extents of DNA strand breakage in these cells. In contrast to the extensive DNA damage induced in control transfectants by 1-h exposure to cytotoxic concentrations of menadione, the extent of DNA breakage detected in GSHPx-rich transfectants was remarkably reduced (6- to 9-fold, p less than 0.005).