Small changes in timing of breeding among subarctic passerines over a 32‐year period

Many bird populations in temperate regions have advanced their timing of breeding in response to a warming climate in recent decades. However, long‐term trends in temperature differ geographically and between seasons, and so do responses of local breeding populations. Data on breeding bird phenology from subarctic and arctic passerine populations are scarce, and relatively little data has been recorded in open‐nesting species. We investigated the timing of breeding and its relationship to spring temperature of 14 mainly open‐nesting passerine species in subarctic Swedish Lapland over a period of 32 years (1984–2015). We estimated timing of breeding from the progress of post‐juvenile moult in mist‐netted birds, a new method exploring the fact that the progress of post‐juvenile moult correlates with age. Although there was a numerical tendency for earlier breeding in most species (on average ?0.09 days/year), changes were statistically significant in only three species (by ?0.16 to ?0.23 days/year). These figures are relatively low compared with what has been found in other long‐term studies but are similar to a few other studies in subarctic areas. Generally, annual hatching dates were negatively correlated with mean temperature in May. This correlation was stronger in long‐distance than in short‐distance migrants. Although annual temperatures at high northern latitudes have increased over recent decades, there was no long‐term increase in mean temperature in May over the study period at this subarctic site. This is probably the main reason why there were only small long‐term changes in hatching dates.     

在韩国境内Potentilla fragarioides var.sprengeliana的遗传多样 …

根据２２个等位酶位点遗传变异,探讨了韩国境内委陵菜（Ｐｏｔｅｎｔｉｌｌａ ｆｒａｇａｒｉｏｉｄｅｓ Ｌ．ｖａｒ．ｓｐｒｅｎｇｅｌｉａｎａ）的遗传多样性和种群结构。酶位点的多态位点百分比为５９．１％。种和种群水平上的遗传多样性比较高,分别为Ｈｅｓ＝０．２１０,Ｈｅｐ＝０．１９９;而种群的分化水平则相对较低（ＧＳＴ＝０．０７４）。１９个种群中随机交配的偏差为ＦＩＳ＝０．３３１。每代迁移数的间接估计     

On the Capacity Losses Seen for Optimized Nano‐Si Composite Electrodes in Li‐Metal Half‐Cells

While the use of silicon‐based electrodes can increase the capacity of Li‐ion batteries considerably, their application is associated with significant capacity losses. In this work, the influences of solid electrolyte interphase (SEI) formation, volume expansion, and lithium trapping are evaluated for two different electrochemical cycling schemes using lithium‐metal half‐cells containing silicon nanoparticle–based composite electrodes. Lithium trapping, caused by incomplete delithiation, is demonstrated to be the main reason for the capacity loss while SEI formation and dissolution affect the accumulated capacity loss due to a decreased coulombic efficiency. The capacity losses can be explained by the increasing lithium concentration in the electrode causing a decreasing lithiation potential and the lithiation cut‐off limit being reached faster. A lithium‐to‐silicon atomic ratio of 3.28 is found for a silicon electrode after 650 cycles using 1200 mAhg^?1 capacity limited cycling. The results further show that the lithiation step is the capacity‐limiting step and that the capacity losses can be minimized by increasing the efficiency of the delithiation step via the inclusion of constant voltage delithiation steps. Lithium trapping due to incomplete delithiation consequently constitutes a very important capacity loss phenomenon for silicon composite electrodes.     

LAPTM4B controls the sphingolipid and ether lipid signature of small extracellular vesicles

Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is a four-membrane spanning ceramide interacting protein that regulates mTORC1 signaling. Here, we show that LAPTM4B is sorted into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) and released in small extracellular vesicles (sEVs) into conditioned cell culture medium and human urine. Efficient sorting of LAPTM4B into ILV membranes depends on its third transmembrane domain containing a sphingolipid interaction motif (SLim). Unbiased lipidomic analysis reveals a strong enrichment of glycosphingolipids in sEVs secreted from LAPTM4B knockout cells and from cells expressing a SLim-deficient LAPTM4B mutant. The altered sphingolipid profile is accompanied by a distinct SLim-dependent co-modulation of ether lipid species. The changes in the lipid composition of sEVs derived from LAPTM4B knockout cells is reflected by an increased stability of membrane nanodomains of sEVs. These results identify LAPTM4B as a determinant of the glycosphingolipid profile and membrane properties of sEVs.     

Construction of chimeric dual-chain avidin by tandem fusion of the related avidins

Background

Avidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences in the tandem gene have limited the use of dual-chain avidin in biotechnological applications.

Principal Findings

To overcome challenges associated with the original dual-chain avidin, we developed chimeric dual-chain avidin, which is a tandem fusion of avidin and avidin-related protein 4 (AVR4), another member of the chicken avidin gene family. We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin. Additionally, PCR amplification of the hybrid gene was more efficient, thus enabling more convenient and straightforward modification of the dual-chain avidin. When studied closer, the generated chimeric dual-chain avidin showed biphasic biotin dissociation.

Significance

The improved dual-chain avidin introduced here increases its potential for future applications. This molecule offers a valuable base for developing bi-functional avidin tools for bioseparation, carrier proteins, and nanoscale adapters. Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.     

Nox4-dependent ROS modulation by amino endoperoxides to induce apoptosis in cancer cells

Tumor metastasis is the main cause of death in cancer patients. Anoikis resistance is one critical malefactor of metastatic cancer cells to resist current clinical chemotherapeutic treatments. Although endoperoxide-containing compounds have long been suggested as anticancer drugs, few have been clinically employed due to their instability, complex synthesis procedure or low tumor cell selectivity. Herein, we describe a one-pot strategy to synthesize novel amino endoperoxides and their derivatives with good yields and stabilities. In vitro cell-based assays revealed that 4 out of the 14 amino endoperoxides selectively induce metastatic breast carcinoma cells but not normal breast cells to undergo apoptosis, in a dose-dependent manner. Mechanistic studies showed that the most potent amino endoperoxide, 4-Me, is selective for cancer cells expressing a high level of Nox4. The anticancer effects are further shown to be associated with reduced O₂⁻:H₂O₂ ratio and increased ·OH level in the cancerous cells. Animal study showed that 4-Me impairs orthotopic breast tumor growth as well as tumor cell metastasis to lymph nodes. Altogether, our study suggests that anticancer strategies that focus on redox-based apoptosis induction in tumors are clinically viable.     

Reduced breeding success of Pied Flycatchers Ficedula hypoleuca along regulated rivers

Most large rivers in northern Sweden are regulated to produce hydropower, with subsequent effects on flow dynamics and aquatic insect communities. Several studies have shown that aquatic and terrestrial systems are intimately connected via the export of emergent aquatic insects, but few have assessed how human modifications of aquatic habitats may influence this connection. We compared breeding success of the insectivorous Pied Flycatcher Ficedula hypoleuca in near‐riparian upland forests along two regulated and two free‐flowing large rivers in northern Sweden over 3 years. The regulated rivers showed lower aquatic insect export to the surroundings, as a consequence of regulation‐induced loss of suitable aquatic insect habitats. Survival of Pied Flycatcher nestlings was 10–15% greater along the free‐flowing rivers. Females breeding near the free‐flowing rivers also started egg‐laying earlier and with greater synchrony than those at the regulated rivers, and showed a smaller decrease in weight during breeding than did females along the regulated rivers. However, there were no differences in occupation rate, clutch size or number of successfully hatched juveniles between regulated and free‐flowing rivers. As regulated rivers showed lower abundance of flying aquatic insects, which may also reduce the abundance of terrestrial invertebrate prey, regulation‐induced changes in the export of emergent aquatic insects may explain both directly and indirectly the observed reduction in Pied Flycatcher breeding success along regulated rivers. Large‐scale river regulation may therefore impair the breeding success of insectivorous birds through impacts on prey availability.     

The Length of the A-M3 Linker Is a Crucial Determinant of the Rate of the Ca2+ Transport Cycle of Sarcoplasmic Reticulum Ca2+-ATPase

Ion translocation by the sarcoplasmic reticulum Ca²⁺-ATPase depends on large movements of the A-domain, but the driving forces have yet to be defined. The A-domain is connected to the ion-binding membranous part of the protein through linker regions. We have determined the functional consequences of changing the length of the linker between the A-domain and transmembrane helix M3 (“A-M3 linker”) by insertion and deletion mutagenesis at two sites. It was feasible to insert as many as 41 residues (polyglycine and glycine-proline loops) in the flexible region of the linker without loss of the ability to react with Ca²⁺ and ATP and to form the phosphorylated Ca₂E1P intermediate, but the rate of the energy-transducing conformational transition to E2P was reduced by >80%. Insertion of a smaller number of residues gave effects gradually increasing with the length of the insertion. Deletion of two residues at the same site, but not replacement with glycine, gave a similar reduction as the longest insertion. Insertion of one or three residues in another part of the A-M3 linker that forms an α-helix (“A3 helix”) in E2/E2P conformations had even more profound effects on the ability of the enzyme to form E2P. These results demonstrate the importance of the length of the A-M3 linker and of the position and integrity of the A3 helix for stabilization of E2P and suggest that, during the normal enzyme cycle, strain of the A-M3 linker could contribute to destabilize the Ca₂E1P state and thereby to drive the transition to E2P.The sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)² is a membrane-bound ion pump that transports Ca²⁺ against a steep concentration gradient, utilizing the energy derived from ATP hydrolysis (1–3). It belongs to the family of P-type ATPases, in which the γ-phosphoryl group of ATP is transferred to a conserved aspartic acid residue during the reaction cycle. Both phospho and dephospho forms of the enzyme undergo transitions between so-called E1 and E2 conformations (Scheme 1). The E1 and E1P states display specificity for reaction with ATP and ADP, respectively (“kinase activity”), whereas E2P and E2 react with water and P_i instead of nucleotide (“phosphatase activity”). The E1 dephosphoenzyme of the Ca²⁺-ATPase binds two Ca²⁺ ions with high affinity from the cytoplasmic side, thereby triggering the phosphorylation from ATP. In E1P, the Ca²⁺ ions are occluded with no access to either side of the membrane, and Ca²⁺ is released to the luminal side after the conformational transition to E2P, likely in exchange for protons being countertransported. The structural organization and domain movements leading to Ca²⁺ translocation have recently been elucidated by crystallization of SERCA in various conformational states thought to represent intermediates in the pump cycle (4–7). SERCA is made up of 10 membrane-spanning mostly helical segments, M1–M10 (numbered from the N terminus), of which M4–M6 and M8 contribute liganding groups for Ca²⁺ binding, and a cytoplasmic headpiece separated into three distinct domains, named A (“actuator”), P (“phosphorylation”), and N (“nucleotide binding”). The A-domain appears to undergo considerable movement during the functional cycle. In the E1/E1P states, the highly conserved TGE¹⁸³S loop of the A-domain is at great distance from the catalytic center containing nucleotide-binding residues and the phosphorylated Asp³⁵¹ of the P-domain, but during the Ca₂E1P → E2P transition, the A-domain rotates ∼90° around an axis perpendicular to the membrane, thereby moving the TGE¹⁸³S loop into close contact with the catalytic site such that Glu¹⁸³ can catalyze dephosphorylation of E2P (8, 9). During the dephosphorylation, Glu¹⁸³ likely coordinates the water molecule attacking the aspartyl phosphoryl bond and withdraws a hydrogen. Hence, the movement of the A-domain during the Ca₂E1P → E2P transition is the event that changes the catalytic specificity from kinase activity to phosphatase activity. During the dephosphorylation of E2P → E2, there is only a slight change of the position of the A-domain, and a large back-rotation is needed to reach the E1 form from E2; thus, the A-domain rotation defines the difference between the E1/E1P class of conformations and the E2/E2P class. Because the A-domain is physically connected to transmembrane helices M1–M3 through the linker segments A-M1, A-M2, and A-M3, the A-domain movement occurring during the Ca₂E1P → E2P transition may be a key event in the opening of the Ca²⁺ sites toward the lumen, thus explaining the coupling of ATP hydrolysis to Ca²⁺ translocation. An important unanswered question is, however, how the movement of the A-domain is brought about. Which are the driving forces that destabilize Ca₂E1P and/or stabilize E2P such that the energy-transducing Ca₂E1P → E2P transition takes place? To answer this, it seems important to elucidate the exact roles of the linkers. Intriguing results have been obtained by Suzuki and co-workers, who demonstrated the importance of the A-M1 linker in connection with luminal release of Ca²⁺ from E2P (10). In this study, we have addressed the role of the A-M3 linker. An alignment of two crystal structures thought to resemble the Ca₂E1P and E2·P_i forms (5), respectively, is shown in Fig. 1. The A-domain rotation is associated with formation of a helix (“A3 helix”) in the N-terminal part of the A-M3 linker, and this helix seems to interact with a helix bundle consisting of the P5–P7 helices of the P-domain, a feature exhibited by all published crystal structures of the E2 type (cf. supplemental Fig. S1 and Ref. 11). Moreover, when structures of similar crystallographic resolution are compared (as in Fig. 1), the non-helical part of the A-M3 linker in E2-type structures has a higher relative temperature factor (“B-factor”) than the corresponding segment in Ca₂E1P (Fig. 1C, thick part colored orange-red for high temperature factor), thus suggesting a higher degree of freedom of movement relative to Ca₂E1P. Hence, the A-M3 linker appears more strained in Ca₂E1P compared with E2 forms, and the greater flexibility of the linker in E2 forms may promote the formation of the A3 helix.Open in a separate windowSCHEME 1.Ca²⁺-ATPase reaction cycle.Open in a separate windowFIGURE 1.A-M3 linker configuration in E1- and E2-type crystal structures. Crystal structures with Protein Data Bank codes 2zbd (Ca₂E1P analog) and 1wpg (E2·P_i analog) are shown aligned. A, overview of structure 2zbd in bluish colors with green A-M3 linker and structure 1wpg in reddish colors with wheat A-M3 linker. B, magnification of the A-M3 linker (corresponding to the red box in A) with arrows indicating site 1, between Glu²⁴³ and Gln²⁴⁴, and site 2, between Gly²³³ and Lys²³⁴, in both conformations. The green A-M3 linker to the right is structure 2zbd. The wheat A-M3 linker to the left is structure 1wpg. Note the kinked A3 helix forming part of the latter structure. C, same A-M3 linker structures as in B but with the magnitude of the temperature factor (B-factor) indicated in colors (red > orange > yellow > green > blue) and by tube diameter. Because the two crystal structures selected here as E1- and E2-type representatives have similar crystallographic resolution (2.40 and 2.30 Å, respectively), the differences in temperature factor in specific regions provide direct information about chain flexibility.Here, we have determined the functional consequences of changing the length (and thereby likely the strain) of the A-M3 linker. Polyglycine and glycine-proline loops of varying lengths were inserted at two different sites in the linker (Fig. 1), and deletions were also studied. Rather unexpectedly, we were able to insert as many as 41 residues in one of the sites without loss of expression or ability to react with Ca²⁺ and ATP, forming Ca₂E1P, but the Ca₂E1P → E2P transition was greatly affected.