A hybrid stochastic-deterministic approach to explore multiple infection and evolution in HIV

To study viral evolutionary processes within patients, mathematical models have been instrumental. Yet, the need for stochastic simulations of minority mutant dynamics can pose computational challenges, especially in heterogeneous systems where very large and very small sub-populations coexist. Here, we describe a hybrid stochastic-deterministic algorithm to simulate mutant evolution in large viral populations, such as acute HIV-1 infection, and further include the multiple infection of cells. We demonstrate that the hybrid method can approximate the fully stochastic dynamics with sufficient accuracy at a fraction of the computational time, and quantify evolutionary end points that cannot be expressed by deterministic models, such as the mutant distribution or the probability of mutant existence at a given infected cell population size. We apply this method to study the role of multiple infection and intracellular interactions among different virus strains (such as complementation and interference) for mutant evolution. Multiple infection is predicted to increase the number of mutants at a given infected cell population size, due to a larger number of infection events. We further find that viral complementation can significantly enhance the spread of disadvantageous mutants, but only in select circumstances: it requires the occurrence of direct cell-to-cell transmission through virological synapses, as well as a substantial fitness disadvantage of the mutant, most likely corresponding to defective virus particles. This, however, likely has strong biological consequences because defective viruses can carry genetic diversity that can be incorporated into functional virus genomes via recombination. Through this mechanism, synaptic transmission in HIV might promote virus evolvability.     

Exonuclease hDIS3L2 specifies an exosome‐independent 3′‐5′ degradation pathway of human cytoplasmic mRNA

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5′‐3′ exoribonuclease hXRN1 and the 3′‐5′ exoribonucleolytic RNA exosome complex. However, in addition to the exosome‐associated 3′‐5′ exonucleases hDIS3 and hDIS3L, the human genome encodes another RNase II/R domain protein—hDIS3L2. Here, we show that hDIS3L2 is an exosome‐independent cytoplasmic mRNA 3′‐5′ exonuclease, which exhibits processive activity on structured RNA substrates in vitro. hDIS3L2 associates with hXRN1 in an RNA‐dependent manner and can, like hXRN1, be found on polysomes. The impact of hDIS3L2 on cytoplasmic RNA metabolism is revealed by an increase in levels of cytoplasmic RNA processing bodies (P‐bodies) upon hDIS3L2 depletion, which also increases half‐lives of investigated mRNAs. Consistently, RNA sequencing (RNA‐seq) analyses demonstrate that depletion of hDIS3L2, like downregulation of hXRN1 and hDIS3L, causes changed levels of multiple mRNAs. We suggest that hDIS3L2 is a key exosome‐independent effector of cytoplasmic mRNA metabolism.     

A heterotypic assembly mechanism regulates CHIP E3 ligase activity

CHIP (C‐terminus of Hsc70‐interacting protein) and its worm ortholog CHN‐1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin‐proteasome system (UPS). CHN‐1 can cooperate with UFD‐2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN‐1–UFD‐2 complex in Caenorhabditis elegans. Our data show that UFD‐2 binding promotes the cooperation between CHN‐1 and ubiquitin‐conjugating E2 enzymes by stabilizing the CHN‐1 U‐box dimer. However, HSP70/HSP‐1 chaperone outcompetes UFD‐2 for CHN‐1 binding, thereby promoting a shift to the autoinhibited CHN‐1 state by acting on a conserved residue in its U‐box domain. The interaction with UFD‐2 enables CHN‐1 to efficiently ubiquitylate and regulate S‐adenosylhomocysteinase (AHCY‐1), a key enzyme in the S‐adenosylmethionine (SAM) regeneration cycle, which is essential for SAM‐dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN‐1 and UFD‐2 in substrate ubiquitylation.     

The evolution of the Aristolochia pallida complex (Aristolochiaceae) challenges traditional taxonomy and reflects large‐scale glacial refugia in the Mediterranean

The taxonomy of the Mediterranean Aristolochia pallida complex has been under debate since several decades with the following species currently recognized: A. pallida, A. lutea, A. nardiana, A. microstoma, A. merxmuelleri, A. croatica, and A. castellana. These taxa are distributed from Iberia to Turkey. To reconstruct phylogenetic and biogeographic patterns, we employed cpDNA sequence variation using both noncoding (intron and spacer) and protein‐coding regions (i.e., trnK intron, matK gene, and trnK‐psbA spacer). Our results show that the morphology‐based traditional taxonomy was not corroborated by our phylogenetic analyses. Aristolochia pallida, A. lutea, A. nardiana, and A. microstoma were not monophyletic. Instead, strong geographic signals were detected. Two major clades, one exclusively occurring in Greece and a second one of pan‐Mediterranean distribution, were found. Several subclades distributed in Greece, NW Turkey, Italy, as well as amphi‐Adriatic subclades, and a subgroup of southern France and Spain, were revealed. The distribution areas of these groups are in close vicinity to hypothesized glacial refugia areas in the Mediterranean. According to molecular clock analyses the diversification of this complex started around 3–3.3 my, before the onset of glaciation cycles, and the further evolution of and within major lineages falls into the Pleistocene. Based on these data, we conclude that the Aristolochia pallida alliance survived in different Mediterranean refugia rarely with low, but often with a high potential for range extension, and a high degree of morphological diversity.     

Adipose‐derived stem cell spheroids are superior to single‐cell suspensions to improve fat autograft long‐term survival

Autologous fat transplantation is a widely used procedure for surgical reconstruction of tissues. The resorption rate of this transplantation remains high and unpredictable, reinforcing the need of adjuvant treatments that increase the long‐term stability of grafts. Adipose‐derived stem cells (ASC) introduced as single cells in fat has been shown clinically to reduce the resorption of fat grafts. On the other hand, the formulation of ASC into cell spheroids results in the enhancement of their regenerative potential. In this study, we developed a novel method to produce highly homogeneous ASC spheroids and characterized their features and efficacy on fat transplantation. Spheroids conserved ASC markers and multipotency. A regenerative gene expression profile was maintained, and genes linked to autophagy were upregulated whereas proliferation was decreased. Their secreted proteome was enriched in comparison with single‐cell ASC suspension. Addition of spheroids to fat graft in an animal model of transplantation resulted in a better graft long‐term stability when compared to single ASC suspension. In conclusion, we provide a novel method to manufacture homogenous ASC spheroids. These ASC spheroids are superior to ASC in single‐cell suspension to improve the stability of fat transplants, reinforcing their potential in reconstructive surgery.     

Geomagnetic information modulates nocturnal migratory restlessness but not fueling in a long distance migratory songbird

Geomagnetic cues have been shown to influence migratory orientation and migratory fuelling in night‐migratory songbird species. Here, we used captive‐bred northern wheatears Oenanthe oenanthe from the southern Norwegian population to show that other aspects of the birds’ migratory program can be influenced by magnetic cues as well. We observed that the amount of migratory restlessness increased strongly with progression of the migratory season when the birds were kept constantly in the magnetic field of northern Germany, but the amount of migratory restlessness decreased when the magnetic field changed along the birds’ natural flyway are simulated. Thus, the Earth's magnetic field can also act as a ‘signpost’ cue for fine‐tuning the spatio‐temporal course of migration.     

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris: functional characterisation of a novel promoter

Applied Microbiology and Biotechnology - As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a...     

Defeating the Warrior: genetic architecture of triticale resistance against a novel aggressive yellow rust race

Key message

Genome-wide association mapping of resistance against the novel, aggressive ‘Warrior’ race of yellow rust in triticale revealed a genetic architecture with some medium-effect QTL and a quantitative component, which in combination confer high levels of resistance on both leaves and ears.

Abstract

Yellow rust is an important destructive fungal disease in small grain cereals and the exotic ‘Warrior’ race has recently conquered Europe. The aim of this study was to investigate the genetic architecture of yellow rust resistance in hexaploid winter triticale as the basis for a successful resistance breeding. To this end, a diverse panel of 919 genotypes was evaluated for yellow rust infection on leaves and ears in multi-location field trials and genotyped by genotyping-by-sequencing as well as for known Yr resistance loci. Genome-wide association mapping identified ten quantitative trait loci (QTL) for yellow rust resistance on the leaves and seven of these also for ear resistance. The total genotypic variance explained by the QTL amounted to 44.0% for leaf and 26.0% for ear resistance. The same three medium-effect QTL were identified for both traits on chromosomes 1B, 2B, and 7B. Interestingly, plants pyramiding the resistance allele of all three medium-effect QTL were generally most resistant, but constitute less than 5% of the investigated triticale breeding material. Nevertheless, a genome-wide prediction yielded a higher predictive ability than prediction based on these three QTL. Taken together, our results show that yellow rust resistance in winter triticale is genetically complex, including both medium-effect QTL as well as a quantitative resistance component. Resistance to the novel ‘Warrior’ race of this fungal pathogen is consequently best achieved by recurrent selection in the field based on identified resistant lines and can potentially be assisted by genomic approaches.

     

Objective detection of apoptosis in rat renal tissue sections using light microscopy and free image analysis software with subsequent machine learning: Detection of apoptosis in renal tissue

Objective

The current study proposes an automated machine learning approach for the quantification of cells in cell death pathways according to DNA fragmentation.

A fully reversible 25-hydroxy steroid kinase involved in oxygen-independent cholesterol side-chain oxidation

The degradation of cholesterol and related steroids by microbes follows fundamentally different strategies in aerobic and anaerobic environments. In anaerobic bacteria, the primary C26 of the isoprenoid side chain is hydroxylated without oxygen via a three-step cascade: (i) water-dependent hydroxylation at the tertiary C25, (ii) ATP-dependent dehydration to form a subterminal alkene, and (iii) water-dependent hydroxylation at the primary C26 to form an allylic alcohol. However, the enzymes involved in the ATP-dependent dehydration have remained unknown. Here, we isolated an ATP-dependent 25-hydroxy-steroid kinase (25-HSK) from the anaerobic bacterium Sterolibacterium denitrificans. This highly active enzyme preferentially phosphorylated the tertiary C25 of steroid alcohols, including metabolites of cholesterol and sitosterol degradation or 25-OH-vitamin D₃. Kinetic data were in agreement with a sequential mechanism via a ternary complex. Remarkably, 25-HSK readily catalyzed the formation of γ-(¹⁸O)₂-ATP from ADP and the C25-(¹⁸O)₂-phosphoester. The observed full reversibility of 25-HSK with an equilibrium constant below one can be rationalized by an unusual high phosphoryl transfer potential of tertiary steroid C25-phosphoesters, which is ≈20 kJ mol⁻¹ higher than that of standard sugar phosphoesters and even slightly greater than the β,γ-phosphoanhydride of ATP. In summary, 25-HSK plays an essential role in anaerobic bacterial degradation of zoo- and phytosterols and shows only little similarity to known phosphotransferases.