Functional and physical competition between phospholamban and its mutants provides insight into the molecular mechanism of gene therapy for heart failure

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLB_M) can compete with wild-type PLB (PLB_W) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLB_W, gain-of-function mutant I40A, or LOF mutants S16E (phosphorylation mimic) or L31A. Inhibitory potency was defined as the fractional increase in K_Ca, measured from the Ca²⁺-dependence of ATPase activity. At saturating PLB, the inhibitory potency of I40A was about three times that of PLB_W, while the potency of each of the LOF PLB_M was about one third that of PLB_W. However, there was no significant variation in the apparent SERCA affinity for these four PLB variants. When SERCA was co-reconstituted with mixtures of PLB_W and LOF PLB_M, inhibitory potency was reduced relative to that of PLB_W alone. Furthermore, FRET between donor-labeled SERCA and acceptor-labeled PLB_W was decreased by both (unlabeled) LOF PLB_M. These results show that LOF PLB_M can compete both physically and functionally with PLB_W, provide a rational explanation for the partial success of S16E-based gene therapy in animal models of heart failure, and establish a powerful platform for designing and testing more effective PLB_M targeted for gene therapy of heart failure in humans.     

Revisión taxonómica del complejo Bauhinia macranthera (Fabaceae: Caesalpinioideae: Cercideae), un grupo endémico del centro y noreste de México

Resumen  La presente revisión tuvo como objetivo la resolución del complejoBauhinia macranthera. Por medio de un estudio de morfología comparada que incluyó polen, se reconocen aBauhinia bartlettii, B. macranthera yB. ramosissima; se restableceB. retifolia y se propone aB. ramosissima var. uniflora, como nueva combinación.

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The purpose of this study was the resolution of theBauhinia macranthera complex using comparative morphology, including pollen.Bauhinia bartlettii, B. macranthera B. ramosissima, andB. retifolia are recognized, andB. ramosissima var. uniflora is proposed as a new combination.

     

Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes

The spatiotemporal dynamics of triglyceride (TG) storage in unilocular adipocytes are not well understood. Here we applied ex vivo technology to study trafficking and metabolism of fluorescent fatty acids in adipose tissue explants. Live imaging revealed multiple cytoplasmic nodules surrounding the large central lipid droplet (cLD) of unilocular adipocytes. Each cytoplasmic nodule harbors a series of closely associated cellular organelles, including micro–lipid droplets (mLDs), mitochondria, and the endoplasmic reticulum. Exogenously added free fatty acids are rapidly adsorbed by mLDs and concurrently get esterified to TG. This process is greatly accelerated by insulin. mLDs transfer their content to the cLD, serving as intermediates that mediate packaging of newly synthesized TG in the large interior of a unilocular adipocyte. This study reveals novel cell biological features that may contribute to the mechanism of adipocyte hypertrophy.     

Identification of a New Borrelia Species among Small Mammals in Areas of Northern Spain Where Lyme Disease Is Endemic

The role of small mammals as reservoir hosts for Borrelia burgdorferi was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by Ixodes ricinus nymphs was found in the small mammal populations studied that correlated with the near-absence of B. burgdorferi sensu lato in 184 animals tested and with the lack of transmission of B. burgdorferi sensu lato to I. ricinus larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable B. burgdorferi sensu lato repertoire (B. burgdorferi sensu stricto, Borrelia garinii, Borrelia valaisiana, and Borrelia afzelii). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other Borrelia species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to B. burgdorferi sensu lato was found in the animal sera, suggesting cross-reactivity between B. burgdorferi sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for B. burgdorferi sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.     

NF‐κB‐dependent secretome of senescent cells can trigger neuroendocrine transdifferentiation of breast cancer cells

Cellular senescence is characterized by a stable proliferation arrest in response to stresses and the acquisition of a senescence‐associated secretory phenotype, called SASP, composed of numerous factors including pro‐inflammatory molecules, proteases, and growth factors. The SASP affects the environment of senescent cells, especially during aging, by inducing and modulating various phenotypes such as paracrine senescence, immune cell activity, and extracellular matrix deposition and organization, which critically impact various pathophysiological situations, including fibrosis and cancer. Here, we uncover a novel paracrine effect of the SASP: the neuroendocrine transdifferentiation (NED) of some epithelial cancer cells, evidenced both in the breast and prostate. Mechanistically, this effect is mediated by NF‐κB‐dependent SASP factors, and leads to an increase in intracellular Ca²⁺ levels. Consistently, buffering Ca²⁺ by overexpressing the CALB1 buffering protein partly reverts SASP‐induced NED, suggesting that the SASP promotes NED through a SASP‐induced Ca²⁺ signaling. Human breast cancer dataset analyses support that NED occurs mainly in p53 WT tumors and in older patients, in line with a role of senescent cells and its secretome, as they are increasing during aging. In conclusion, our work, uncovering SASP‐induced NED in some cancer cells, paves the way for future studies aiming at better understanding the functional link between senescent cell accumulation during aging, NED and clinical patient outcome.     

Human mucin MUC1 RNA undergoes different types of alternative splicing resulting in multiple isoforms

MUC1 is a transmembrane mucin with important functions in normal and transformed cells, carried out by the extracellular domain or the cytoplasmic tail. A characteristic feature of the MUC1 extracellular domain is the variable number of tandem repeats (VNTR) region. Alternative splicing may regulate MUC1 expression and possibly function. We developed an RT-PCR method for efficient isolation of MUC1 mRNA isoforms that allowed us to evaluate the extent of alternative splicing of MUC1 and elucidate some of the rules that govern this process. We cloned and analyzed 21, 24, and 36 isoforms from human tumor cell lines HeLa, MCF7, and Jurkat, respectively, and 16 from normal activated human T cells. Among the 78 MUC1 isoforms we isolated, 76 are new and different cells showed varied MUC1 expression patterns. The VNTR region of exon 2 was recognized as an intron with a fixed 5′ splice site but variable 3′ splice sites. We also report that the 3506 A/G SNP in exon 2 can regulate 3′ splice sites selection in intron 1 and produce different MUC1 short isoform proteins. Furthermore, the SNP A to G mutation was also observed in vivo, during de novo tumor formation in MUC1^+/?Kras^G12D/+Pten^loxP/loxP mice. No specific functions have been associated with previously reported short isoforms. We now report that one new G SNP-associated isoform MUC1/Y-LSP, but not the A SNP-associated isoform MUC1/Y, inhibits tumor growth in immunocompetent but not immunocompromised mice.     

Recombinant protein production in cultures of an Escherichia coli trp − strain

Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp ^– derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-l batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l. Correspondence to: F. Bolivar     

Silicon acquisition by bananas (c.V. Grande Naine) is increased in presence of the arbuscular mycorrhizal fungus <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> MUCL 41833

Aim

This work aimed to investigate the role of arbuscular mycorrhizal fungi (AMF) in the uptake and accumulation of silicon (Si) in banana plants. Si is recognized as a significant element that helps plants resist stresses.

Methods

A pot experiment compared the growth, Si and P accumulation of banana plants pre-colonized or not by an AMF and exposed or not to Si added to the growth substrate.

Results

A marked increase in Si was noticed in pseudostem, leaves and roots of pre-colonized banana plants, in presence as well as in absence of Si added to the growth substrate. Without Si addition, this accumulation was 60 % and 45 % higher in pseudostem and leaves, respectively, while it was 47 % and 41 % in presence of Si added to the substrate. In roots, this increase was 23 % and 52 % in presence and absence of Si added to the substrate, respectively. Phosphorus content in shoots and roots was likewise significantly increased in presence of AMF or Si.

Conclusion

Our findings revealed that pre-colonized banana plants accumulated more Si in shoot and roots than non-mycorrhizal plants and may thus represent a potential novel avenue to explore banana resistance to pests and diseases.

     

Molecular dynamics,thermodynamic, and mutational binding studies for tumor-specific LyP-1 in complex with p32

Recent studies in tumor homing peptides have shown the specificity of LyP-1 (CGNKRTRGC) to tumor lymphatics. In this present work, we evaluated the possible interactions between cyclic LyP-1 and its receptor, p32, with molecular dynamics and docking studies in order to lead the design of novel LyP-1 derivatives, which could bind to p32 more effectively and perform enhanced antitumor effect. The total binding enthalpy energies have been obtained by MM-PBSA thermodynamic computations and the favorability of p32.LyP-1 complex in water has been shown by explicit water MD computations. The last 30 ns of molecular dynamics trajectory have shown the strong interaction of LyP-1 with the inner surface chains of p32, especially with chains B and C. ALA-SCAN mutagenesis studies have indicated the considerable influence of Asn3, Lys4, Arg5, and Arg7 amino acid residues on the specific binding of LyP-1. Within the knowledge of the critical role of p32 receptor in cancer cell metabolism, this study can lead to further developments in anticancer therapy by targeting p32 with LyP-1 derivatives as active targeting moiety. This data can also be applied for the development of new drug delivery systems in which LyP-1 can be used for its targeting and anticancer properties.