Isoform Specificity of the Na/K-ATPase Association and Regulation by Phospholemman

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction. FRET was measured as the increase in the donor fluorescence (CFP-NKA-α1 or CFP-NKA-α2) during progressive acceptor (PLM-YFP) photobleach in HEK-293 cells. Both pairs exhibited robust FRET (maximum of 23.6 ± 3.4% for NKA-α1 and 27.5 ± 2.5% for NKA-α2). Donor fluorescence depended linearly on acceptor fluorescence, indicating a 1:1 PLM:NKA stoichiometry for both isoforms. PLM phosphorylation induced by cAMP-dependent protein kinase and protein kinase C activation drastically reduced the FRET with both NKA isoforms. However, submaximal cAMP-dependent protein kinase activation had less effect on PLM-NKA-α2 versus PLM-NKA-α1. Surprisingly, ouabain virtually abolished NKA-PLM FRET but only partially reduced co-immunoprecipitation. PLM-CFP also showed FRET to PLM-YFP, but the relationship during progressive photobleach was highly nonlinear, indicating oligomers involving ≥3 monomers. Using cardiac myocytes from wild-type mice and mice where NKA-α1 is ouabain-sensitive and NKA-α2 is ouabain-resistant, we assessed the effects of PLM phosphorylation on NKA-α1 and NKA-α2 function. Isoproterenol enhanced internal Na⁺ affinity of both isoforms (K_½ decreased from 18.1 ± 2.0 to 11.5 ± 1.9 mm for NKA-α1 and from 16.4 ± 2.5 to 10.4 ± 1.5 mm for NKA-α2) without altering maximum transport rate (V_max). Protein kinase C activation also decreased K_½ for both NKA-α1 and NKA-α2 (to 9.4 ± 1.0 and 9.1 ± 1.1 mm, respectively) but increased V_max only for NKA-α2 (1.9 ± 0.4 versus 1.2 ± 0.5 mm/min). In conclusion, PLM associates with and modulates both NKA-α1 and NKA-α2 in a comparable but not identical manner.Cardiac Na/K-ATPase (NKA)³ regulates intracellular Na⁺, which in turn affects intracellular Ca²⁺ and contractility via Na⁺/Ca²⁺ exchange. Members of the FXYD family of small, single membrane-spanning proteins, including phospholemman (PLM) and the NKA γ-subunit (1), have emerged recently as tissue-specific regulators of NKA. PLM is the only FXYD protein known to be highly expressed in cardiac myocytes and is also unique within the family in that it is phosphorylated at two or more sites by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) (2, 3). In the heart, PLM is a major phosphorylation target for both PKA and PKC.Co-immunoprecipitation experiments have demonstrated that PLM is physically associated with NKA (4–8), and this is not affected by PLM phosphorylation (6, 7). We have shown recently (9) that PLM and NKA are in very close proximity, such that fluorescence resonance energy transfer (FRET) occurs. PLM phosphorylation by either PKA or PKC reduces the FRET significantly, suggesting that although PLM and NKA are not physically dissociated upon phosphorylation, their interaction is altered. PLM inhibits NKA (4, 8, 10, 11), mostly by reducing the affinity of the pump for internal Na⁺. PLM phosphorylation relieves this inhibition and thus mediates the enhancement of NKA function by α- and β-adrenergic stimulation in mouse ventricular myocytes (10, 11).There are multiple NKA isoforms in cardiac myocytes. NKA-α1 is the dominant, ubiquitous isoform, whereas NKA-α2 and NKA-α3 are present in relatively small amounts and in a species-dependent manner (12). For instance, the adult rodent heart expresses NKA-α1 and NKA-α2, although dogs and monkeys do not have the NKA-α2 subunit (13). In humans all three NKA-α isoforms can be detected (14). It has been suggested that NKA-α2 and NKA-α3 are located mainly in the T-tubules, at the junctions with the sarcoplasmic reticulum, where they could regulate local Na⁺/Ca²⁺ exchange and thus cardiac myocyte Ca²⁺. There is rather convincing evidence supporting such a model in the smooth muscle (15). However, things are less clear in the heart. The functional density of NKA-α2 is significantly higher in the T-tubules (versus external sarcolemma) in cardiac myocytes from both rats (16, 17) and mice (18), but their precise localization with respect to the junctions with the sarcoplasmic reticulum is not known. Based on Ca²⁺ transients from heterozygous NKA-α1^+/− and NKA-α2^+/− mice, James et al. (19) concluded that NKA-α2 is involved in cardiac myocyte Ca²⁺ regulation, whereas NKA-α1 is not. Further support for this idea came from the observation that replacing mouse NKA-α2 with a low affinity mutant leads to a loss of glycoside inotropy (20), and increased expression of NKA-α2 decreased the Na⁺/Ca²⁺ exchange current and Ca²⁺ transients (21). However, other findings challenge the preferential role of NKA-α2 in regulating intracellular Ca²⁺ and contractility. Moseley et al. (22) showed that NKA-α1^+/− mice were severely compromised, and Dostanic et al. (23) showed that NKA-α1 is also physically and functionally associated with the Na⁺/Ca²⁺ exchanger.In this context, it is important to determine whether NKA-α1 and NKA-α2 interact differently with PLM. The data available so far on this are contradictory. We have found (7) that NKA-α1, NKA-α2, and NKA-α3 isoforms co-immunoprecipitate PLM, both unphosphorylated and phosphorylated, in rabbit heart. In contrast, Silverman et al. (8) reported that NKA-α1 but not NKA-α2 co-immunoprecipitate with PLM in ventricular myocytes from guinea pig. The functional data are also contradictory. PLM was found to reduce the affinity for Na⁺ of both NKA-α1 and NKA-α2 isoforms in a heterologous expression system (4), whereas Silverman et al. (8) reported that forskolin-induced PLM phosphorylation results in a higher NKA-α1-mediated current and no change in the current generated by NKA-α2.Here we used two methods to investigate whether the interaction and functional effects of PLM on NKA are NKA-α isoform-specific. First, we used FRET to assess the interaction between PLM-YFP and CFP-NKA-α1/CFP-NKA-α2 transfected in HEK-293 cells and how PLM phosphorylation by PKA and PKC affects this interaction. Second, we measured NKA function in myocytes isolated from wild-type (WT) mice and mice where NKA isoforms have swapped ouabain affinities (SWAP; NKA-α1 is ouabain-sensitive, whereas NKA-α2 is ouabain-resistant) (23). In this way we could test the effect of β-adrenergic stimulation separately on NKA-α1 and NKA-α2 isoforms in the native myocyte environment, as an indicator of the functional interaction with PLM. Our results indicate that NKA-α1 and NKA-α2 interact similarly with PLM, and this interaction is equally affected by PLM phosphorylation.     

Species turnover at small scales in dune slack plant communities

Patterns of both species accumulation with increasing area and of individual species occurrences depend on the scale level considered. This study investigated community diversity and individual species turnover patterns between four scale levels within 2×2 m² nested plots situated in a dune slack plant community. The number of species increased with plot area following a log–log function, with a slope of 0.23. However, species turnover was higher between the lowest scale levels, indicating limitations on species occurrences at the 25×25 cm² scale level. Alpha diversity in rectangular plots was significantly higher than in square plots of the same area. There were strong differences between individual species turnover patterns. Most species occurrence patterns had a box-counting fractal dimension value between 0.8 and 1.6, which is rather low compared with other studies on larger scale levels. Analyses of occurrence probabilities and scale area plots showed that there is a systematic deviation from self-similarity at the smallest scale level. Species had a lower frequency than expected from a fractal distribution, suggesting a higher level of species aggregation. The higher species diversity turnover at the smallest scale level can be linked to a higher spatial aggregation of individual species, due to biotic or abiotic limitations on their occurrence. These results confirm the general nature of the pattern of break-down of self-similarity at the smallest scale level considered.

Zusammenfassung

Sowohl das Muster des Artenanstiegs mit zunehmender Fläche als auch das Muster des Auftretens einzelner Arten hängen vom betrachteten Skalenlevel ab. Diese Studie untersuchte die Diversität der Lebensgemeinschaft und die Muster der Fluktuationen einzelner Arten auf vier Skalenlevels innerhalb von 2×2 m² ineinander geschachtelten Versuchsflächen in einer Pflanzengemeinschaft der Dünentäler. Die Zahl der Arten nahm mit der Versuchsfläche entsprechend einer log–log Funktion mit einer Steigung von 0.23 zu. Die Artenfluktuation zwischen den niedrigsten Skalenlevels war jedoch größer und weist darauf hin, dass es Limitierungen für das Auftreten der Arten auf dem 25×25 cm² Skalenlevel gibt. Die Alpha-Diversität war in rechteckigen Versuchsflächen signifikant größer als in quadratischen Versuchsflächen der gleichen Größe. Es gab größe Unterschiede in den Mustern der Fluktuation einzelner Arten. Die meisten Muster des Auftretens der Arten hatten fraktale Box-Counting-Dimensions-Werte zwischen 0.8 und 1.6, was relativ gering im Vergleich zu Studien auf größeren Skalenlevels ist. Die Analysen der Auftretenswahrscheinlichkeit und der Probefläche der Skalenlevels zeigten, dass es eine systematische Abweichung von der Selbstähnlichkeit auf dem kleinsten Skalenlevel gibt. Die Arten hatten eine geringere Häufigkeit als die aufgrund einer fraktalen Verteilung erwartete, was einen höheren Level der Artaggregation vermuten lässt. Die größere Fluktuation der Artendiversität auf dem kleinsten Skalenlevel kann mit einer größeren räumlichen Aggregation einzelner Arten aufgrund von biotischen und abiotischen Beschränkungen ihres Vorkommens in Verbindung gebracht werden. Diese Ergebnisse bestätigen die generelle Natur des Musters des Zusammenbruchs der Selbstähnlichkeit auf den kleinsten betrachteten Skalenlevels.     

Archaeobatrachian paraphyly and pangaean diversification of crown-group frogs

Current models for the early diversification of living frogs inferred from morphological, ontogenetic, or DNA sequence data invoke very different scenarios of character evolution and biogeography. To explore central controversies on the phylogeny of Anura, we analyzed nearly 4000 base pairs of mitochondrial and nuclear DNA for the major frog lineages. Likelihood-based analyses of this data set are congruent with morphological evidence in supporting a paraphyletic arrangement of archaeobatrachian frogs, with an (Ascaphus + Leiopelma) clade as the sister-group of all other living anurans. The stability of this outcome is reinforced by screening for phylogenetic bias resulting from site-specific rate variation, homoplasy, or the obligatory use of distantly related outgroups. Twenty-one alternative branching and rooting hypotheses were evaluated using a nonparametric multicomparison test and parametric bootstrapping. Relaxed molecular clock estimates situate the emergence of crown-group anurans in the Triassic, approximately 55 million years prior to their first appearance in the fossil record. The existence of at least four extant frog lineages on the supercontinent Pangaea before its breakup gains support from the estimation that three early splits between Laurasia- and Gondwana-associated families coincide with the initial rifting of these landmasses. This observation outlines the potential significance of this breakup event in the formation of separate Mesozoic faunal assemblages in both hemispheres.     

Essential role for CD40 ligand interactions in T lymphocyte-mediated modulation of the murine immune response to pneumococcal capsular polysaccharides

Protection against infection with pneumococci is provided by anti-capsular polysaccharide (caps-PS) Abs. We investigated whether CD40 ligand (CD40L) plays a role in T lymphocyte-mediated regulation of the immune response to caps-PS, which are considered thymus-independent Ags. Administration of MR1, an antagonist mAb against murine CD40L, in BALB/c mice immunized with Pneumovax resulted in an inhibition of the IgM and IgG Ab response for various caps-PS serotypes. Evidence for the involvement of CD4(+) T lymphocytes in the Ab response to caps-PS was obtained in SCID/SCID mice that, when reconstituted with B lymphocytes and CD4(+) T lymphocytes, mounted a higher specific IgM response compared with SCID/SCID mice reconstituted with only B lymphocytes. This helper effect of CD4(+) T lymphocytes was abrogated by MR1. Blocking CD40L in vitro decreased the IgM response to caps-PS and abolished the helper effect of CD4(+) T lymphocytes. CD8(+) T lymphocyte-depleted murine spleen cells mounted a higher in vivo immune response than total murine spleen cells, which provided evidence for a suppressive role of CD8(+) T lymphocytes on the anti-caps-PS immune response. CD4(+) T lymphocyte-depleted murine spleen cells, leaving a B and CD8(+) T lymphocyte fraction, elicited only a weak in vivo and in vitro Ab response, which was enhanced after MR1 administration. In summary, our data provide evidence that T lymphocytes contribute to the regulation of the anti-caps-PS immune response in a CD40L-dependent manner.     

Familial mental retardation syndrome ATR-16 due to an inherited cryptic subtelomeric translocation, t(3;16)(q29;p13.3)

In the search for genetic causes of mental retardation, we have studied a five-generation family that includes 10 individuals in generations IV and V who are affected with mild-to-moderate mental retardation and mild, nonspecific dysmorphic features. The disease is inherited in a seemingly autosomal dominant fashion with reduced penetrance. The pedigree is unusual because of (1) its size and (2) the fact that individuals with the disease appear only in the last two generations, which is suggestive of anticipation. Standard clinical and laboratory screening protocols and extended cytogenetic analysis, including the use of high-resolution karyotyping and multiplex FISH (M-FISH), could not reveal the cause of the mental retardation. Therefore, a whole-genome scan was performed, by linkage analysis, with microsatellite markers. The phenotype was linked to chromosome 16p13.3, and, unexpectedly, a deletion of a part of 16pter was demonstrated in patients, similar to the deletion observed in patients with ATR-16 syndrome. Subsequent FISH analysis demonstrated that patients inherited a duplication of terminal 3q in addition to the deletion of 16p. FISH analysis of obligate carriers revealed that a balanced translocation between the terminal parts of 16p and 3q segregated in this family. This case reinforces the role of cryptic (cytogenetically invisible) subtelomeric translocations in mental retardation, which is estimated by others to be implicated in 5%-10% of cases.     

Regional mapping of a liver alpha-subunit gene of phosphorylase kinase (PHKA) to the distal region of human chromosome Xp.

X-linked liver glycogenosis (XLG) is a glycogen storage disorder resulting from deficient activity of phosphorylase kinase (PHK). PHK consists of four different subunits: alpha, beta, gamma, and delta. Several genes encoding PHK subunits have been cloned and localized, but only the muscle alpha-subunit (PHKA) gene has been assigned to the X chromosome, in the region Xq12----q13. However, we have previously excluded the muscle PHKA gene as a candidate gene for the XLG mutation, as linkage analysis indicated that the mutation responsible for XLG is located in Xp22 and not in Xq12----q13. We report here the chromosomal localization by in situ hybridization of a liver PHKA gene to the distal region of chromosome Xp. Strong hybridization signals were observed on the distal part of the short arm of a chromosome identified as the X chromosome by cohybridization with an X chromosome-specific centromeric probe. The localization of this gene in the same chromosomal region as the disease gene responsible for XLG suggests that the liver PHKA gene is a highly likely candidate gene for the XLG mutation.     

The PRISMA 2020 statement: An updated guideline for reporting systematic reviews

Matthew Page and co-authors describe PRISMA 2020, an updated reporting guideline for systematic reviews and meta-analyses.

The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement, published in 2009, was designed to help systematic reviewers transparently report why the review was done, what the authors did, and what they found. Over the past decade, advances in systematic review methodology and terminology have necessitated an update to the guideline. The PRISMA 2020 statement replaces the 2009 statement and includes new reporting guidance that reflects advances in methods to identify, select, appraise, and synthesise studies. The structure and presentation of the items have been modified to facilitate implementation. In this article, we present the PRISMA 2020 27-item checklist, an expanded checklist that details reporting recommendations for each item, the PRISMA 2020 abstract checklist, and the revised flow diagrams for original and updated reviews.

Systematic reviews serve many critical roles. They can provide syntheses of the state of knowledge in a field, from which future research priorities can be identified; they can address questions that otherwise could not be answered by individual studies; they can identify problems in primary research that should be rectified in future studies; and they can generate or evaluate theories about how or why phenomena occur. Systematic reviews therefore generate various types of knowledge for different users of reviews (such as patients, healthcare providers, researchers, and policy makers).[1,2]To ensure a systematic review is valuable to users, authors should prepare a transparent, complete, and accurate account of why the review was done, what they did (such as how studies were identified and selected) and what they found (such as characteristics of contributing studies and results of meta-analyses). Up-to-date reporting guidance facilitates authors achieving this.[3]The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement published in 2009 (hereafter referred to as PRISMA 2009)[4–10] is a reporting guideline designed to address poor reporting of systematic reviews.[11] The PRISMA 2009 statement comprised a checklist of 27 items recommended for reporting in systematic reviews and an “explanation and elaboration” paper[12–16] providing additional reporting guidance for each item, along with exemplars of reporting. The recommendations have been widely endorsed and adopted, as evidenced by its co-publication in multiple journals, citation in over 60 000 reports (Scopus, August 2020), endorsement from almost 200 journals and systematic review organisations, and adoption in various disciplines. Evidence from observational studies suggests that use of the PRISMA 2009 statement is associated with more complete reporting of systematic reviews,[17–20] although more could be done to improve adherence to the guideline.[21]Many innovations in the conduct of systematic reviews have occurred since publication of the PRISMA 2009 statement. For example, technological advances have enabled the use of natural language processing and machine learning to identify relevant evidence,[22–24] methods have been proposed to synthesise and present findings when meta-analysis is not possible or appropriate,[25–27] and new methods have been developed to assess the risk of bias in results of included studies.[28,29] Evidence on sources of bias in systematic reviews has accrued, culminating in the development of new tools to appraise the conduct of systematic reviews.[30, 31] Terminology used to describe particular review processes has also evolved, as in the shift from assessing “quality” to assessing “certainty” in the body of evidence.[32] In addition, the publishing landscape has transformed, with multiple avenues now available for registering and disseminating systematic review protocols,[33, 34] disseminating reports of systematic reviews, and sharing data and materials, such as preprint servers and publicly accessible repositories. To capture these advances in the reporting of systematic reviews necessitated an update to the PRISMA 2009 statement.Summary points

• To ensure a systematic review is valuable to users, authors should prepare a transparent, complete, and accurate account of why the review was done, what they did, and what they found
• The PRISMA 2020 statement provides updated reporting guidance for systematic reviews that reflects advances in methods to identify, select, appraise, and synthesise studies
• The PRISMA 2020 statement consists of a 27-item checklist, an expanded checklist that details reporting recommendations for each item, the PRISMA 2020 abstract checklist, and revised flow diagrams for original and updated reviews
• We anticipate that the PRISMA 2020 statement will benefit authors, editors, and peer reviewers of systematic reviews, and different users of reviews, including guideline developers, policy makers, healthcare providers, patients, and other stakeholders