Time-resolved spectroscopy of the primary photosynthetic processes of membrane-bound reaction centers from an antenna-deficient mutant of Rhodobacter capsulatus

The primary photosynthetic reactions in whole membranes of the antenna-deficient mutant strain U43 (pTXA6–10) of Rhodobacter capsulatus are studied by transient absorption and emission spectroscopy with subpicosecond time resolution. Extensive similarities between the transient absorption data on whole membranes and on isolated reaction centers support the idea that the primary processes in isolated reaction centers are not modified by the isolation procedure

Metabolic engineering of murine cytotoxic T cells by solute carrier Slc2a1/GLUT1 overexpression to enhance anti-tumor activity

In the last decade, adoptive T-Cell therapy (ACT) has emerged as a successful treatment of hematological malignancies. However, solid tumors pose challenges created by various factors, including poor infiltration, immunosuppressive factors, and the lack of nutrient viability. In combination with the low vascularisation, the nutrient-deprived tumor microenvironment (TME) is mainly created by the elevated aerobic glycolysis in tumor cells. As one of the main precursor metabolites, glucose is used in vast amounts by the tumor, therefore causing a massive concentration drop in the surrounding matrix. The increased surface expression of facilitative glucose transporters from the solute carrier family 2 (GLUT1/Slc2) on tumor cells plays an essential role in this process, giving them a selective advantage in the created TME. Infiltrating T cells would face a deserted tumor landscape that heavily interferes with their metabolism and consequently, their metabolic need to be functionally active could not be fulfilled. This new paradigm of immune escape mechanism has long been ignored but was more and more shifted into the spotlight, recently. Herein, we propose a novel strategy for ACT of metabolically engineered cytotoxic CD8+ T cells to adapt immune cells to the prevalent glucose concentrations in the TME. In this study, we could reveal that the ectopic overexpression of the glucose transporter GLUT1 encoded by Slc2a1 increased the fitness of primary murine CD8+ T cells (CD8+Slc2a1) in hypoglycemic conditions. Additionally, our results showed augmented functional activity and anti-tumor efficacy, in vitro and partially in vivo. We observed CD8+Slc2a1 undergo a changed metabolic reprogramming affecting their transcriptional landscape, oxidative state, and memory formation. These findings set the foundation for future studies on ACT in combination with GLUT1 overexpression in pre-clinical settings.Im letzten Jahrzehnt hat sich Adoptive T-Zell Therapy (ACT) als erfolgreiche Therapie gegen Blutkrebserkranungen bewährt. Jedoch bergen solide Krebsarten eine größere Herausforderung, welche durch die schlechte Immunzellinfiltration, immunsuppressive Faktoren und nicht zuletzt dem Mangel an Nährstoffen definiert werden. In Kombination mit der schlechten Vaskularisierung kreieren Tumorzellen ein nährstoffarmes Tumormikromillieu (TME) durch ihre hochregulierte glykolytische Aktivität. Als eine der wichtigsten Grundmetabolite wird Glukose in großen Mengen vom Tumor entzogen, wodurch ein substanzieller Konzentrationsabfall in der umliegenden Matrix folgt. Die Überexpression des Glukosetransporters GLUT1/SLC2A1 an der Oberfläche der Tumorzellen spielt eine zentrale Rolle in diesem Prozess, indem es diesen einen selektiven Vorteil im entstandenen TME verleiht. Einwandernde T-Zellen sind daher mit einer nährstoffverlassenen Tumorlandschaft konfrontiert, welche signifikant ihren Stoffwechsel beeinträchtigt und folglich ihre metabolischen Bedürfnisse nicht erfüllen kann. Dieses neue Paradigma der Immunflucht würde lange ignoriert aber findet mehr und mehr Beachtung in diesem Kontext. In dieser Arbeit präsentieren wir eine neue Strategie der ACT indem wir metabolisch modifizierte zytotoxische T-Zellen an die vorherrschenden Glukosekonzentrationen des TME anpassen. Wir konnten zeigen, dass die ektope Überexpression des Glukosetransporters GLUT1 (codiert in Slc2a1) die Fitness von primären murinen CD8+ T-Zellen (CD8+Slc2a1) in hypoglykämischen Bedingungen erhöht. Unsere in vitro und in vivo Ergebnisse zeigen zusätzlich eine erhöhte funktionelle Aktivität und Zeichen verbesserter anti-tumor Effektivität. Außerdem konnten wir beobachten, dass CD8+Slc2a1 Zellen eine metabolische Reprogrammierung durchlaufen, welche zur Veränderung des transkriptionellen und oxidativen Zustandes führt und die Gedächtnisbildung beeinflusst. Diese Ergebnisse sollen als einen Grundstein für künftige präklinische Studien der ACT in Kombination mit GLUT1 Überexpression dienen

Detailed studies of the subpicosecond kinetics in the primary electron transfer of reaction centers of Rhodopseudomonas viridis

The primary, light-induced charge separation in reaction centers of Rhodopseudomonas viridis is investigated with femtosecond time resolution. The absorption changes after direct excitation of the primary donor P at 955 nm are investigated in the time range from 100 fs to 600 ps. The experimental data, taken at various probing wavelengths, reveal one subpicosecond and two picosecond time constants: 0.65 ± 0.2 ps, 3.5 ± 0.4 ps, and 200 ± 20 ps. The previously undetected 0.65 ps kinetics can be observed clearly in the spectral range of the Qx and Qy transitions of the monomeric bacteriochlorophylls. The experimental data support the idea that the accessory bacteriochlorophyll B A participates in the electron-transfer process. Reference

Role of tyrosine M210 in the initial charge separation of reaction centers of Rhodobacter sphaeroides

Femtosecond spectroscopy was used in combination with site-directed mutagenesis to study the influence of tyrosine M210 (YM210) on the primary electron transfer in the reaction center of Rhodobacter sphaeroides. The exchange of YM210 to phenylalanine caused the time constant of primary electron transfer to increase from 3.5 f 0.4 ps to 16 f 6 ps while the exchange to leucine increased the time constant even more to 22 f 8 ps. The results suggest that tyrosine M210 is important for the fast rate of the primary electron transfer

First known records of the specialist bee, Colletes aestivalis (Hymenoptera: Colletidae), in an urban area

Here we report on the first known records of the Heuchera specialist bee Colletes aestivalis in an urban area. This bee was found by a participant in a citizen science project in a residential garden in a suburb that abuts St. Louis, Missouri. The females were found only visiting Heuchera richardsonii, and the males were seen scouting for females by hovering over the plants. This finding shows that urban areas can support uncommon specialist bees and the utility of engaging non-experts in cataloging biodiversity

Temperature dependence of the primary electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides

The primary electron transfer (ET) in reaction centers (RC) of Rhodobacter sphaeroides is investigated as a function of temperature with femtosecond time resolution. For temperatures from 300 to 25 K the ET to the bacteriopheophytin is characterized by a biphasic time dependence. The two time constants of τ1=3.5±0.4 ps and τ2=1.2±0.3 ps at T=300 K decrease continously with temperature to values of τ1=1.4±0.3 ps and τ2=0.3±0.15 ps at 25 K. The experimental results indicate that the ET is not thermally activated and that the same ET mechanisms are active at room and low temperatures. All observations are readily rationalized by a two-step ET model with the monomeric bacteriochlorophyll as a real electron carrier

Gain-of-Sensitivity Mutations in a Trim5-Resistant Primary Isolate of Pathogenic SIV Identify Two Independent Conserved Determinants of Trim5α Specificity

Retroviral capsid recognition by Trim5 blocks productive infection. Rhesus macaques harbor three functionally distinct Trim5 alleles: Trim5αQ, Trim5αTFP and Trim5CypA. Despite the high degree of amino acid identity between Trim5αQ and Trim5αTFP alleles, the Q/TFP polymorphism results in the differential restriction of some primate lentiviruses, suggesting these alleles differ in how they engage these capsids. Simian immunodeficiency virus of rhesus macaques (SIVmac) evolved to resist all three alleles. Thus, SIVmac provides a unique opportunity to study a virus in the context of the Trim5 repertoire that drove its evolution in vivo. We exploited the evolved rhesus Trim5α resistance of this capsid to identify gain-of-sensitivity mutations that distinguish targets between the Trim5αQ and Trim5αTFP alleles. While both alleles recognize the capsid surface, Trim5αQ and Trim5αTFP alleles differed in their ability to restrict a panel of capsid chimeras and single amino acid substitutions. When mapped onto the structure of the SIVmac239 capsid N-terminal domain, single amino acid substitutions affecting both alleles mapped to the β-hairpin. Given that none of the substitutions affected Trim5αQ alone, and the fact that the β-hairpin is conserved among retroviral capsids, we propose that the β-hairpin is a molecular pattern widely exploited by Trim5α proteins. Mutations specifically affecting rhesus Trim5αTFP (without affecting Trim5αQ) surround a site of conservation unique to primate lentiviruses, overlapping the CPSF6 binding site. We believe targeting this site is an evolutionary innovation driven specifically by the emergence of primate lentiviruses in Africa during the last 12 million years. This modularity in targeting may be a general feature of Trim5 evolution, permitting different regions of the PRYSPRY domain to evolve independent interactions with capsid

Phylogeny and History of the Lost SIV from Crab-Eating Macaques: SIVmfa

In the 20th century, thirteen distinct human immunodeficiency viruses emerged following independent cross-species transmission events involving simian immunodeficiency viruses (SIV) from African primates. In the late 1900s, pathogenic SIV strains also emerged in the United Sates among captive Asian macaque species following their unintentional infection with SIV from African sooty mangabeys (SIVsmm). Since their discovery in the 1980s, SIVs from rhesus macaques (SIVmac) and pig-tailed macaques (SIVmne) have become invaluable models for studying HIV pathogenesis, vaccine design and the emergence of viruses. SIV isolates from captive crab-eating macaques (SIVmfa) were initially described but lost prior to any detailed molecular and genetic characterization. In order to infer the origins of the lost SIVmfa lineage, we located archived material and colony records, recovered its genomic sequence by PCR, and assessed its phylogenetic relationship to other SIV strains. We conclude that SIVmfa is the product of two cross-species transmission events. The first was the established transmission of SIVsmm to rhesus macaques, which occurred at the California National Primate Research Center in the late 1960s and the virus later emerged as SIVmac. In a second event, SIVmac was transmitted to crab-eating macaques, likely at the Laboratory for Experimental Medicine and Surgery in Primates in the early 1970s, and it was later spread to the New England Primate Research Center colony in 1973 and eventually isolated in 1986. Our analysis suggests that SIVmac had already emerged by the early 1970s and had begun to diverge into distinct lineages. Furthermore, our findings suggest that pathogenic SIV strains may have been more widely distributed than previously appreciated, raising the possibility that additional isolates may await discovery