Biology subjects

Karolak, M.

Publications and source records attributed to Karolak, M..

4 recordsLinked to original sources

bioRxiv2025

Unravelling the intraspecific variation in drought responses in seedlings of European black pine (Pinus nigra J.F. Arnold)

O_LIUnderstanding intraspecific variation in drought tolerance is essential for predicting the adaptive capacity of forest species under climate change. Yet, the molecular basis of this variation remains poorly understood in ecologically and economically important conifers. C_LIO_LIWe integrated high-throughput phenotyping with metabolomics and transcriptomics under standardized soil drying to investigate drought responses across nine climatically distinct provenances of the conifer Pinus nigra. We tested whether drought tolerance--measured as decline in maximum quantum yield of the photosystem II (Fv/Fm)--varies among provenances, follows a climatic cline, and involves trade-off with growth. To identify the underlying molecular basis, we performed metabolomics and transcriptomics in four provenances representing contrasting drought tolerance. C_LIO_LIDrought tolerance varied significantly among provenances and was decoupled from growth, yet showed no differentiation along the climatic cline. Drought tolerant provenances differed from sensitive ones in both constitutive and drought-induced levels of flavonoid and diterpene metabolites. Transcriptomic profiles further highlighted provenance-specific differences in gene expression related to flavonoids. C_LIO_LIOur results demonstrate the utility of integrating automated phenotyping with molecular profiling to uncover the metabolic basis of drought adaptation, laying the groundwork for targeted studies on metabolite function and tolerance strategies in non-model conifers. C_LI

plant biology↗

bioRxiv2025

Combined Deletion of miR-27a and miR-27b Enhances Protection Against Diet-Induced Obesity

MicroRNAs (miRNAs) are frequently encoded within polycistronic clusters thought to function as coherent regulatory units, yet whether individual cluster members act cooperatively or exert distinct physiological effects remains unclear. miR-27a and miR-27b are closely related miRNAs located within separate paralogous miR-23/27/24 clusters, but their in vivo relationship is unresolved. Using isoform-specific knockout models generated by precise CRISPR-based excision of individual miRNA hairpins, we defined how miR-27a and miR-27b regulate adipose thermogenesis and systemic metabolism. High-fat feeding increased miR-27a/b expression and suppressed thermogenic gene programs in subcutaneous white adipose tissue. In primary beige and brown adipocytes, loss of either isoform produced modest activation of thermogenic programs, whereas combined deletion caused additive increases in Ucp1 expression and mitochondrial DNA content. Ex vivo heat production was significantly elevated in double-knockout adipose explants relative to wild-type and single-knockout controls. In vivo, double-knockout mice exhibited increased energy expenditure and, under high-fat diet conditions, reduced adiposity with improved glucose homeostasis compared with wild-type and single-knockout mice. In contrast to metabolic dysfunction reported following whole-cluster deletion, selective miR-27 loss reveals a protective thermogenic program. These findings demonstrate that miRNA clusters need not act as coherent regulatory modules and identify miR-27a/b as a cooperative thermogenic checkpoint with therapeutic implications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/666011v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@7d6b74org.highwire.dtl.DTLVardef@199d56org.highwire.dtl.DTLVardef@1a87e48org.highwire.dtl.DTLVardef@fab51a_HPS_FORMAT_FIGEXP M_FIG C_FIG * CRISPR isoform-specific knockouts isolate miR-27a/b contributions to metabolism. *Dual miR-27a/b loss drives cooperative thermogenic activation. *Double knockouts enhance energy expenditure and metabolic fitness in diet-induced obesity. *miR-27a/b deletion reveals that clustered miRNAs can exert non-coherent regulatory roles.

physiology↗

bioRxiv2025

Exosome Trafficking Is a Key Regulator of Adipocyte Thermogenesis

Activation of beige adipocytes enhances energy expenditure and promotes metabolic health, presenting a promising approach for combating obesity and diabetes. As part of this process, thermogenesis, fueled in part by uncoupled mitochondrial respiration, plays a central role in converting calories into thermal energy, thereby preventing their storage as fat. Here, we identify a role for exosome trafficking as an intrinsic regulator of beige adipocyte thermogenesis. Exosomes are small extracellular vesicles that mediate cell-cell and intracellular communication by transporting regulatory cargo, including microRNAs, proteins, and lipids. Using both human cells and mouse models, we show that thermogenic activation of beige adipocytes promotes the rapid release of exosomes enriched in microRNAs known to suppress thermogenic programs. Genetic or pharmacological blockade of exosome secretion attenuates thermogenesis, whereas enhancing exosome release amplifies thermogenic output. Mice deficient in the exosome secretion regulator Rab27a exhibit reduced energy expenditure in response to both cold exposure and {beta}3-adrenergic stimulation. These findings establish exosome trafficking as a key contributor to beige adipocyte thermogenic capacity, highlighting an intracellular mechanism that may be leveraged to enhance energy expenditure and treat obesity-related metabolic diseases. Significance StatementThermogenic adipocytes, including beige fat cells, help maintain energy balance by converting excess nutrients into heat, thereby reducing fat storage and supporting metabolic function. Although these cells are known to promote energy expenditure, the intracellular processes that enable their full thermogenic response are not well defined. Here, we show that exosome secretion is required for beige adipocytes to reach their full thermogenic potential. Blocking exosome release dampens this response, while boosting it amplifies thermogenic output. These findings point to exosome release as an essential part of thermogenic regulation and a potential target for improving metabolic health.

cell biology↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗