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Freshwater Assembly Experiment (Exudates 2024) - Jackrel Lab

ABSTRACT: With the intent to understand the drivers of microbial assembly, we used phytoplankton as a model host organism. The external metabolome of thirty-one strains of freshwater algae were collected to better understand the drivers of interactions between phytoplankton hosts and bacteria. To collect the metabolite profile, sterile monocultures of the 31 phytoplankton strains were grown under identical conditions. When each strain reached carrying capacity, the culture was filtered through a 0.22um filter, removing all phytoplankton cells. This filtrate was then acidified to a pH of 2.5 and stored at 4C for 2-29 hours, until it could be processed through the solid phase extraction column. To prep the column, 1 cartridge volume (1mL) of 100% methanol was passed through the column. Once the column was prepped, 50mL of cell-free, acidified culture was passed through the column to collect the organic material. The column was cleaned with 2 cartridge volume of 0.01M HCl and air dried for 5 minutes. The sample was eluted into a deep-well plate over dry ice with 1mL of 100% methanol. The plates were stored at -80C until ready for processing.

INSTRUMENT(S): Orbitrap Exploris 240

ORGANISM(S): Chlamydomonas Asymmetrica (ncbitaxon:51683) Tetradesmus Lagerheimii Ncbitaxon:3075448 Ophiocytium Majus (ncbitaxon:73019) Chlorella Vulgaris (ncbitaxon:3077) Selenastrum Capricornutum (ncbitaxon:118073) Microcystis Aeruginosa (ncbitaxon:1126) Mesotaenium Caldariorum (ncbitaxon:31321) Chlamydomonas Reinhardtii (ncbitaxon:3055) Gloeocapsa Alpicola (ncbitaxon:1357543) Chlamydomonas Sphaeroides (ncbitaxon:28458) Axilococcus Clingmanii Ncbitaxon:2511175 Parachlorella Kessleri (ncbitaxon:3074) Porphyridium Aerugineum (ncbitaxon:2792) Ettlia Oleoabundans (ncbitaxon:1127754) Chlorella Sorokiniana (ncbitaxon:3076) Desmonostoc Muscorum (ncbitaxon:1179) Vischeria Vischeri Ncbitaxon:2772652 Coelastrum Microporum (ncbitaxon:55409) Nannochloris (ncbitaxon:3187) Monodopsis Ncbitaxon:1745946 Cosmarium Botrytis (ncbitaxon:33101)

SUBMITTER: Sara Jackrel

PROVIDER: MSV000096773 | MassIVE | Fri Jan 03 12:57:00 GMT 2025

REPOSITORIES: MassIVE

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Project description:Marine phytoplankton are a diverse group of photoautotrophic organisms and key mediators in the global carbon cycle. Phytoplankton physiology and biomass accumulation are closely tied to mixed layer depth, but the intracellular metabolic pathways activated in response to changing mixed layer depths remain unexplored. Here, metatranscriptomics was used to characterize the phytoplankton community response to a mixed layer shallowing from 233 meters to 5 meters over the course of two days during the late spring in the Northwest Atlantic. Most phytoplankton genera downregulated core photosynthesis, carbon storage, and carbon fixation genes as the system transitioned from a deep to a shallow mixed layer and shifted towards catabolism of stored carbon ic pathways supportive of rapid cell growth. In contrast, phytoplankton genera exhibited divergent transcriptional strategies for photosystem light harvesting complex genes during this transition. Active infection, taken as the ratio of virus to host transcripts, increased in the Bacillariophyta (diatom) phylum and decreased in the Chlorophyta (green algae) phylum upon mixed layer shallowing. A conceptual model is proposed to provide ecophysiological context for our findings, in which light limitation during deep mixing induces populations into a transcriptional state which maximizes interrupts the oscillating levels of transcripts related to photosynthesis, carbon storage, and carbon fixation found in shallow mixed layers with relatively higher growth rates. We propose that upon sensing high light levels during mixed layer shallowing, phytoplankton resume diel oscillation of core sets of genes enabling photoprotection, biosynthesis and cell replication. Our findings highlight the shared and unique transcriptional response strategies within phytoplankton communities acclimating to the dynamic light environment associated with transient deep mixing and shallowing events during the annual North Atlantic bloom.

Project description:Abstract: Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MP) of the peripheral nervous system (PNS) are main cellular agent controlling this process. Some evidences suggest that resident PNS-MP along with MP of hematogenous origin may be involved but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporters mice, and coherent anti-stoke raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatio-temporal choreography of resident and recently recruited MP after injury and unveiled distinct functions of these subsets with recruited MP responsible of efficient myelin stripping and clearance while resident MP were involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases. Methods: (relevant for this GEO dataset): RNAseq of sciatic nerve macrophages from mice after CCI: Sorted cells were lysed in 100µl of RA1/TCEP buffer (NucleoSpin RNA XS, Macherey-Nagel), snap frozen in liquid nitrogen and stored at -80C until RNA extraction. All samples were processed in parallel and RNA extraction (without Carrier RNA but with on-column DNase treatment) was performed according to manufacturer's instructions (NucleoSpin RNA XS, Macherey-Nagel). RNA was eluted in RNAse-free water. Preparation of cDNA libraries for RNAseq was done using the SmartSeq method according to manufacturer's instructions (SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing, Clontech/TaKaRa). Due to the low number of cells, the total amount of eluted RNA was used as starting material for reverse transcription, followed by 18 cycles of pre-amplification. 1ng of cDNA were used for RNAseq sequencing library preparation, according to manufacturer's instructions (Nextera XT DNA Library Preparation, Illumina). Final samples pooled library prep were sequenced on a Nextseq 500 ILLUMINA with MidOutPut cartridge (2x130Millions of 75 bases reads) with 2 runs (4plex and 5plex), corresponding to 2x30Millions of (paired-end) reads per sample after demultiplexing.

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Freshwater Assembly Experiment (Exudates 2024) - Jackrel Lab

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