Early detection and surveillance of SARS-CoV-2 genomic variants in wastewater using COJAC

The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK

Early detection and surveillance of SARS-CoV-2 genomic variants in wastewater using COJAC

The continuing emergence of SARS-CoV-2 variants of concern and variants of interest emphasizes the need for early detection and epidemiological surveillance of novel variants. We used genomic sequencing of 122 wastewater samples from three locations in Switzerland to monitor the local spread of B.1.1.7 (Alpha), B.1.351 (Beta) and P.1 (Gamma) variants of SARS-CoV-2 at a population level. We devised a bioinformatics method named COJAC (Co-Occurrence adJusted Analysis and Calling) that uses read pairs carrying multiple variant-specific signature mutations as a robust indicator of low-frequency variants. Application of COJAC revealed that a local outbreak of the Alpha variant in two Swiss cities was observable in wastewater up to 13 d before being first reported in clinical samples. We further confirmed the ability of COJAC to detect emerging variants early for the Delta variant by analysing an additional 1,339 wastewater samples. While sequencing data of single wastewater samples provide limited precision for the quantification of relative prevalence of a variant, we show that replicate and close-meshed longitudinal sequencing allow for robust estimation not only of the local prevalence but also of the transmission fitness advantage of any variant. We conclude that genomic sequencing and our computational analysis can provide population-level estimates of prevalence and fitness of emerging variants from wastewater samples earlier and on the basis of substantially fewer samples than from clinical samples. Our framework is being routinely used in large national projects in Switzerland and the UK.</p

A systems-wide understanding of photosynthetic acclimation in algae and higher plants

The ability of phototrophs to colonise different environments relied on the robust protection against oxidative stress in phototrophs, a critical requirement for the successful evolutionary transition from water to land. Photosynthetic organisms have developed numerous strategies to adapt their photosynthetic apparatus to changing light conditions in order to optimise their photosynthetic yield, crucial for life to exist on Earth. Photosynthetic acclimation is an excellent example of the complexity of biological systems, in which highly diverse processes, ranging from electron excitation over protein protonation to enzymatic processes coupling ion gradients with biosynthetic activity interact on drastically different timescales, ranging from picoseconds to hours. An efficient functioning of the photosynthetic apparatus and its protection is paramount for efficient downstream processes including metabolism and growth. Modern experimental techniques can be successfully integrated with theoretical and mathematical models to promote our understanding of underlying mechanisms and principles. This Review aims to provide a retrospective analysis of multidisciplinary photosynthetic acclimation research carried out by members of the Marie Curie Initial Training Project “AccliPhot”, placing the results in a wider context. The Review also highlights the applicability of photosynthetic organisms for industry, particularly with regards to the cultivation of microalgae. It aims to demonstrate how theoretical concepts can successfully complement experimental studies broadening our knowledge of common principles in acclimation processes in photosynthetic organisms, as well as in the field of applied microalgal biotechnology

Identification and characterization of chloroplast protein phosphatases involved in regulation of photosynthesis in Chlamydomonas reinhardtii

The aim of this thesis was to provide novel information about the regulation of photosynthetic electron transfer in the green alga Chlamydomonas reinhardtii, with a focus on the regulation of thylakoid protein phosphorylation. In Arabidopsis, two antagonist pairs of protein kinases and phosphates are known. The kinase STN7 and the phosphatase PPH1 act on light-harvesting antennae proteins in regulating state transitions, while the kinase STN8 and the phosphatase PBCP act on PSII core subunits. In this project, the thylakoid kinase/phosphatase network in Chlamydomonas is explored. As yet, in Chlamydomonas only the STN7 homologue, STT7, was known. Within this work three chloroplast phosphatases regulating the photosynthetic electron transfer chain are newly identified and characterized: CrPPH1, CrPBCP and CrPBCP-LIKE. The thesis begins with a general introduction on the photosynthetic electron chain and its regulation, with a particular focus on the role of thylakoid protein phosphorylation and the known kinases and phosphatases involved. Phosphorylation of the light-harvesting antenna regulates the allocation of light energy to the two photosystems, in a process known as state transitions. Phosphorylation of the light-harvesting antenna favours its binding to PSI (state 2) while its de-phosphorylation favours its association with PSII (state 1). In the first chapter of the results the roles of CrPPH1 and CrPBCP are investigated, specifically during state transitions. Like its homologue in Arabidospis, CrPPH1 is required for efficient de-phosphorylation of the light-harvesting antenna and transition from state 2 to state 1. However CrPBCP is also involved in these process, unlike PBCP in Arabidopsis. In Chlamydomonas the two phosphatases have partly redundant roles, reflected in the partial overlap of their substrate specificities for the subunits of the light-harvesting antenna. The second chapter of the results presents the characterization of CrPBCP-LIKE, specifically under high light. This phosphatase, which is closely related to CrPBCP, is not required for state transitions, but plays a role in acclimation to high light and photoprotection. The third chapter of the results begins with a review of the biochemical approaches used to study the structural reorganization of the thylakoid membrane protein complexes during light acclimation processes, with a discussion of some technical issues. An optimized protocol is presented, to isolate thylakoid membranes from Chlamydomonas while preserving the phosphorylation state of thylakoid proteins and to study thylakoid supercomplex organization. Our results indicate that the roles of PPH1 and particularly PBCP homologs in Chlamydomonas are different from those of the plant counterparts, and a different scenario for the regulation of LHCII phosphorylation and the state transition mechanism appear in the green algae. The thesis ends with a general discussion and all the results are put together in a wider perspective

Phosphorylation of the Light-Harvesting Complex II Isoform Lhcb2 Is Central to State Transitions

Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (>98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis

Regulation of Light Harvesting in Chlamydomonas reinhardtii Two Protein Phosphatases Are Involved in State Transitions

International audienceProtein phosphorylation plays important roles in short-term regulation of photosynthetic electron transfer, and during state transitions, the kinase STATE TRANSITION7 (STT7) ofChlamydomonas reinhardtiiphosphorylates components of light-harvesting antenna complex II (LHCII). This reversible phosphorylation governs the dynamic allocation of a part of LHCII to PSI or PSII, depending on light conditions and metabolic demands, but counteracting phosphatase(s) remain unknown inC. reinhardtii. Here we analyzed state transitions inC. reinhardtiimutants of two phosphatases, PROTEIN PHOSPHATASE1 and PHOTOSYSTEM II PHOSPHATASE, which are homologous to proteins that antagonize the state transition kinases (STN7 and STN8) in Arabidopsis (Arabidopsis thaliana). The transition from state 2 to state 1 was retarded inpph1, and surprisingly also inpbcp. However, both mutants eventually returned to state 1. In contrast, the double mutantpph1;pbcpappeared strongly locked in state 2. The complex phosphorylation patterns of the LHCII trimers and of the monomeric subunits were affected in the phosphatase mutants. Their analysis indicated that the two phosphatases have different yet overlapping sets of protein targets. The dual control of thylakoid protein dephosphorylation and the more complex antenna phosphorylation patterns inC. reinhardtiicompared to Arabidopsis are discussed in the context of the stronger amplitude of state transitions and the more diverse LHCII isoforms in the alga

Wastewater monitoring outperforms case numbers as a tool to track COVID-19 incidence dynamics when test positivity rates are high

Wastewater-based epidemiology (WBE) has been shown to coincide with, or anticipate, confirmed COVID-19 case numbers. During periods with high test positivity rates, however, case numbers may be underreported, whereas wastewater does not suffer from this limitation. Here we investigated how the dynamics of new COVID-19 infections estimated based on wastewater monitoring or confirmed cases compare to true COVID-19 incidence dynamics. We focused on the first pandemic wave in Switzerland (February to April, 2020), when test positivity ranged up to 26%. SARS-CoV-2 RNA loads were determined 2–4 times per week in three Swiss wastewater treatment plants (Lugano, Lausanne and Zurich). Wastewater and case data were combined with a shedding load distribution and an infection-to-case confirmation delay distribution, respectively, to estimate infection incidence dynamics. Finally, the estimates were compared to reference incidence dynamics determined by a validated compartmental model. Incidence dynamics estimated based on wastewater data were found to better track the timing and shape of the reference infection peak compared to estimates based on confirmed cases. In contrast, case confirmations provided a better estimate of the subsequent decline in infections. Under a regime of high-test positivity rates, WBE thus provides critical information that is complementary to clinical data to monitor the pandemic trajectory

Vitamin D pathway gene polymorphisms affecting daclatasvir plasma concentration at 2 weeks and 1 month of therapy

Vitamin D (VD) influences genetic expression through its receptor (VDR). VD pathway gene polymorphisms seem to influence antiviral drug pharmacokinetics and therapeutic outcome/toxicity. We investigated the association between daclatasvir (DCV) plasma concentrations and genetic variants (SNPs) associated with the VD pathway