Supplying the trip to antibody production—nutrients, signaling, and the programming of cellular metabolism in the mature B lineage

The COVID pandemic has refreshed and expanded recognition of the vital role that sustained antibody (Ab) secretion plays in our immune defenses against microbes and of the importance of vaccines that elicit Ab protection against infection. With this backdrop, it is especially timely to review aspects of the molecular programming that govern how the cells that secrete Abs arise, persist, and meet the challenge of secreting vast amounts of these glycoproteins. Whereas plasmablasts and plasma cells (PCs) are the primary sources of secreted Abs, the process leading to the existence of these cell types starts with naive B lymphocytes that proliferate and differentiate toward several potential fates. At each step, cells reside in specific microenvironments in which they not only receive signals from cytokines and other cell surface receptors but also draw on the interstitium for nutrients. Nutrients in turn influence flux through intermediary metabolism and sensor enzymes that regulate gene transcription, translation, and metabolism. This review will focus on nutrient supply and how sensor mechanisms influence distinct cellular stages that lead to PCs and their adaptations as factories dedicated to Ab secretion. Salient findings of this group and others, sometimes exhibiting differences, will be summarized with regard to the journey to a distinctive metabolic program in PCs.

Encoding stereochemical molecular information on cyclophanes using non-directional interactions and achiral building blocks

The stereoselective assembly of achiral constituents through a single spontaneous process into complex covalent architectures bearing multiple stereogenic elements currently seems out of reach to the synthetic chemist. It even seems beyond what nature itself has managed to attain through evolution. Here, we show that such an extreme level of control can be achieved by molecular programming, i.e. by implementing stereo-electronic information on synthetic organic building blocks and exploiting the features of the covalent reactions and interactions, whose interplay acts as a powerful assembling algorithm. Remarkably, we show that non-directional bonds and interactions can reliably transfer this information, delivering in near to physiological conditions, high-molecular weight macrocyclic species carrying up to 8-bits of conformational and configuration information. Beyond the field of supramolecular chemistry, this proof of concept should stimulate the on-demand production of highly structured polyfunctional architectures.

Heterogeneity and molecular programming of progenitors for motor neurons and oligodendrocytes

The pMN domain is a restricted domain in the ventral spinal cords, defined by the expression of olig2 gene. The fate determination of pMN progenitors is highly temporally and spatially regulated, with motor neurons and oligodendrocyte progenitor cells (OPCs) developing sequentially. Insight into the heterogeneity and molecular programs of pMN progenitors is currently lacking. With the zebrafish model, we identified multiple states of neural progenitors using single-cell sequencing: proliferating progenitors, common progenitors for both motor neurons and OPCs, and restricted precursors for either motor neurons or OPCs. We found specific molecular programs for neural progenitor fate transition, and manipulations of representative genes in the motor neuron or OPC lineage confirmed their critical role in cell fate determination. The transcription factor NPAS3 is necessary for the development of the OPC lineage and can interact with many known genes associated with schizophrenia. Deciphering progenitor heterogeneity and molecular mechanisms for these transitions will elucidate the formation of complex neuron-glia networks in the central nervous system during development, and understand the basis of neurodevelopmental disorders.

Molecular programming in utero modulates hepatic lipid metabolism and adult metabolic risk in obese mother offspring in a sex-specific manner

Male and female offspring of obese mothers are known to differ significantly in their metabolic adaptation and later development of complications. We investigated the sex-dependent responses in obese offspring of mice with maternal obesity, focusing on changes in liver glucose and lipid metabolism. Maternal obesity prior to and during gestation led to hepatic insulin resistance and inflammation in male offspring, while female offspring were protected. These sex differences were explained by more efficient transcriptional and posttranscriptional reprogramming of metabolic pathways to prevent the damaging effects of maternal obesity in females compared to males. These differences were sustained later in life, resulting in a better metabolic balance in female offspring. In conclusion, sex and maternal obesity drive transcriptional and posttranscriptional regulation of major metabolic processes in offspring liver differently, explaining the sexual dimorphism in obesity-associated metabolic risk.

Underpinning the molecular programming attributing heat stress associated thermotolerance in tea (Camellia sinensis (L.) O. Kuntze)

The most daunting issue of global climate change is the deleterious impact of extreme temperatures on tea productivity and quality, which has resulted in a quest among researchers and growers. The current study aims to unravel molecular programming underpinning thermotolerance by characterizing heat tolerance and sensitivity response in 20 tea cultivars. The significantly higher negative influence of heat stress was recorded in a sensitive cultivar with reduced water retention (47%), chlorophyll content (33.79%), oxidation potential (32.48%), and increase in membrane damage (76.4%). Transcriptional profiling of most tolerant and sensitive cultivars identified 78 differentially expressed unigenes with chaperon domains, including low and high molecular weight heat shock protein (HSP) and heat shock transcription factors (HSFs) involved in heat shock response (HSR). Further, predicted transcriptional interactome network revealed their key role in thermotolerance via well-co-ordinated transcriptional regulation of aquaporins, starch metabolism, chlorophyll biosynthesis, calcium, and ethylene mediated plant signaling system. The study identified the key role of HSPs (CsHSP90) in regulating HSR in tea, wherein, structure-based molecular docking revealed the inhibitory role of geldanamycin (GDA) on CsHSP90 by blocking ATP binding site at N-terminal domain of predicted structure. Subsequently, GDA mediated leaf disc inhibitor assay further affirmed enhanced HSR with higher expression of CsHSP17.6, CsHSP70, HSP101, and CsHSFA2 genes in tea. Through the current study, efforts were made to extrapolate a deeper understanding of chaperons mediated regulation of HSR attributing thermotolerance in tea.

Molecular Programming of Drought-Challenged Trichoderma harzianum-Bioprimed Rice (Oryza sativa L.)

Trichoderma biopriming enhances rice growth in drought-stressed soils by triggering various plant metabolic pathways related to antioxidative defense, secondary metabolites, and hormonal upregulation. In the present study, transcriptomic analysis of rice cultivar IR64 bioprimed with Trichoderma harzianum under drought stress was carried out in comparison with drought-stressed samples using next-generation sequencing techniques. Out of the 2,506 significant (p < 0.05) differentially expressed genes (DEGs), 337 (15%) were exclusively expressed in drought-stressed plants, 382 (15%) were expressed in T. harzianum-treated drought-stressed plants, and 1,787 (70%) were commonly expressed. Furthermore, comparative analysis of upregulated and downregulated genes under stressed conditions showed that 1,053 genes (42%) were upregulated and 733 genes (29%) were downregulated in T. harzianum-treated drought-stressed rice plants. The genes exclusively expressed in T. harzianum-treated drought-stressed plants were mostly photosynthetic and antioxidative such as plastocyanin, small chain of Rubisco, PSI subunit Q, PSII subunit PSBY, osmoproteins, proline-rich protein, aquaporins, stress-enhanced proteins, and chaperonins. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis states that the most enriched pathways were metabolic (38%) followed by pathways involved in the synthesis of secondary metabolites (25%), carbon metabolism (6%), phenyl propanoid (7%), and glutathione metabolism (3%). Some of the genes were selected for validation using real-time PCR which showed consistent expression as RNA-Seq data. Furthermore, to establish host–T. harzianum interaction, transcriptome analysis of Trichoderma was also carried out. The Gene Ontology (GO) analysis of T. harzianum transcriptome suggested that the annotated genes are functionally related to carbohydrate binding module, glycoside hydrolase, GMC oxidoreductase, and trehalase and were mainly upregulated, playing an important role in establishing the mycelia colonization of rice roots and its growth. Overall, it can be concluded that T. harzianum biopriming delays drought stress in rice cultivars by a multitude of molecular programming.

Cross-Inhibitor: a time-sensitive molecular circuit based on DNA strand displacement

Designing biochemical systems that can be effectively used in diverse fields, including diagnostics, molecular computing and nanomachines, has long been recognized as an important goal of molecular programming and DNA nanotechnology. A key issue in the development of such practical devices on the nanoscale lies in the development of biochemical components with information-processing capacity. In this article, we propose a molecular device that utilizes DNA strand displacement networks and allows interactive inhibition between two input signals; thus, it is termed a cross-inhibitor. More specifically, the device supplies each input signal with a processor such that the processing of one input signal will interdict the signal of the other. Biochemical experiments are conducted to analyze the interdiction performance with regard to effectiveness, stability and controllability. To illustrate its feasibility, a biochemical framework grounded in this mechanism is presented to determine the winner of a tic-tac-toe game. Our results highlight the potential for DNA strand displacement cascades to act as signal controllers and event triggers to endow molecular systems with the capability of controlling and detecting events and signals.

The transcription factor Pou3f1 provides a new map to the glutamatergic neurons of the cerebellar nuclei

Pou3f1 is a transcription factor involved in early neural differentiation. Cap Analysis Gene Expression (5’-CAGE) analysis reveals that Pou3f1 transcript is highly enriched in the developing cerebellum. Between embryonic (E) days E10.5 and E12.5, Pou3f1 expression is present prominently along the subpial stream (SS), suggesting that Pou3f1+ cells are glutamatergic cerebellar nuclear (CN) neurons. This finding was confirmed by immunofluorescent (IF) co-labeling of Pou3f1 and Atoh1, the master regulator of cells from the rhombic lip (RL) that are destined for neurons of the glutamatergic lineage, as well as in Atoh1-null tissues, in which Pou3f1 expression is absent. Interestingly, the expression of Pax6, another key molecule for CN neuron survival, does not co-localize with that of Pou3f1. In the Pax6-null Small Eye (Sey) mutant, which is characterized by a loss of many glutamatergic CN neurons, Pou3f1+ CN neurons are still present. Furthermore, Pou3f1-labeled cells do not co-express Tbr1, a well-established marker of glutamatergic CN neurons. These results highlight that Pou3f1+ cells are a distinct and previously unrecognized subtype of glutamatergic CN neurons that do not have the “canonical” sequence of Atoh1→Pax6→Tbr1 expressions. Instead, they express Atoh1, Pou3f1, and other markers of CN neurons, Brn2 and Irx3. These findings illustrate that glutamatergic CN neurons that arise from the RL are composed of molecularly heterogeneous subpopulations that are determined by at least two distinct transcriptional programs.Significance StatementThe present work has identified Pou3f1 as a marker for a previously unidentified subtype of glutamatergic cerebellar nuclear neurons, the principal output neurons of the cerebellum. The classical model of glutamatergic CN neurons development follows the sequential expression of transcription factors Atoh1→Pax6→Tbr1. However, we found that the development of Pou3f1+ neurons requires Atoh1 but not Pax6. Moreover, Pou3f1+ neurons do not express Tbr1, but instead express two other transcription factors, Brn2 and Irx3. Anatomically, Pou3f1+ CN neurons populate the interposed and dentate nuclei, while the Tbr1+ CN neurons populate the fastigial nucleus. These findings reveal the heterogeneity of CN neuron populations and the diversity of molecular programming that lead to different CN neuron subtypes.

Isothermal digital detection of microRNAs using background-free molecular circuit

MicroRNAs, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA detection method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design a DNA circuit that converts, thresholds, amplifies, and reports the presence of a specific microRNA, down to the femtomolar concentration. Using a leak absorption mechanism, we were able to suppress nonspecific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNAs: By partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.