DNA-DISK: Automated end-to-end data storage via enzymatic single-nucleotide DNA synthesis and sequencing on digital microfluidics.

In the age of information explosion, the exponential growth of digital data far exceeds the capacity of current mainstream storage media. DNA is emerging as a promising alternative due to its higher storage density, longer retention time, and lower power consumption. To date, commercially mature DNA synthesis and sequencing technologies allow for writing and reading of information on DNA with customization and convenience at the research level. However, under the disconnected and nonspecialized mode, DNA data storage encounters practical challenges, including susceptibility to errors, long storage latency, resource-intensive requirements, and elevated information security risks. Herein, we introduce a platform named DNA-DISK that seamlessly streamlined DNA synthesis, storage, and sequencing on digital microfluidics coupled with a tabletop device for automated end-to-end information storage. The single-nucleotide enzymatic DNA synthesis with biocapping strategy is utilized, offering an ecofriendly and cost-effective approach for data writing. A DNA encapsulation using thermo-responsive agarose is developed for on-chip solidification, not only eliminating data clutter but also preventing DNA degradation. Pyrosequencing is employed for in situ and accurate data reading. As a proof of concept, DNA-DISK successfully stored and retrieved a musical sheet file (228 bits) with lower write-to-read latency (4.4 min of latency per bit) as well as superior automation compared to other platforms, demonstrating its potential to evolve into a DNA Hard Disk Drive in the future.

Digital microfluidics-based digital counting of single-cell copy number variation (dd-scCNV Seq).

Single-cell copy number variations (CNVs), major dynamic changes in humans, result in differential levels of gene expression and account for adaptive traits or underlying disease. Single-cell sequencing is needed to reveal these CNVs but has been hindered by single-cell whole-genome amplification (scWGA) bias, leading to inaccurate gene copy number counting. In addition, most of the current scWGA methods are labor intensive, time-consuming, and expensive with limited wide application. Here, we report a unique single-cell whole-genome library preparation approach based on digital microfluidics for digital counting of single-cell Copy Number Variation (dd-scCNV Seq). dd-scCNV Seq directly fragments the original single-cell DNA and uses these fragments as templates for amplification. These reduplicative fragments can be filtered computationally to generate the original partitioned unique identified fragments, thereby enabling digital counting of copy number variation. dd-scCNV Seq showed an increase in uniformity in the single-molecule data, leading to more accurate CNV patterns compared to other methods with low-depth sequencing. Benefiting from digital microfluidics, dd-scCNV Seq allows automated liquid handling, precise single-cell isolation, and high-efficiency and low-cost genome library preparation. dd-scCNV Seq will accelerate biological discovery by enabling accurate profiling of copy number variations at single-cell resolution.

Synergetic collision and space separation in microfluidic chip for efficient affinity-discriminated molecular selection.

Efficient molecular selection is a prerequisite for generating molecular tools used in diagnosis, pathology, vaccinology, and therapeutics. Selection efficiency is thermodynamically highly dependent on the dissociation equilibrium that can be reached in a single round. Extreme shifting of equilibrium towards dissociation favors the retention of high-affinity ligands over those with lower affinity, thus improving the selection efficiency. We propose to synergize dual effects by deterministic lateral-displacement microfluidics, including the collision-based force effect and the two-dimensional (2D) separation-based concentration effect, to greatly shift the equilibrium. Compared with previous approaches, this system can remove more low- or moderate-affinity ligands and maintain most high-affinity ligands, thereby improving affinity discrimination in selection. This strategy is demonstrated on phage display in both experiment and simulation, and two peptides against tumor markers ephrin type-A receptor 2 (EphA2) and CD71 were obtained with high affinity and specificity within a single round of selection, which offers a promising direction for discovery of robust binding ligands for a wide range of biomedical applications.

Tunable Surface Wettability via Terahertz Electrowave Controlled Vicinal Subnanoscale Water Layer.

Achieving timely, reversible, and long-range remote tunability over surface wettability is highly demanded across diverse fields, including nanofluidic systems, drug delivery, and heterogeneous catalysis. Herein, using molecular dynamic simulations, we show, for the first time, a theoretical design of electrowetting to achieve remotely controllable surface wettability via using a terahertz wave. The key idea driving the design is the unique terahertz collective vibration identified in the vicinal subnanoscale water layer, which is absent in bulk water, enabling efficient energy transfer from the terahertz wave to the rotational motion of the vicinal subnanoscale water layer. Consequently, a frequency-specific alternating terahertz electric field near the critical strength can significantly affect the local hydrogen-bonding network of the contact water layer on the solid surface, thereby achieving tunable surface wettability.

Genome-wide identification and expression analysis of the SPL transcription factor family and its response to abiotic stress in Pisum sativum L.

Squamous promoter binding protein-like (SPL) genes encode plant-specific transcription factors (TFs) that play essential roles in modulating plant growth, development, and stress response. Pea (Pisum sativum L.) is a coarse grain crop of great importance in food production, biodiversity conservation and molecular genetic research, providing genetic information and nutritional resources for improving agricultural production and promoting human health. However, only limited researches on the structure and functions of SPL genes exist in pea (PsSPLs). In this study, we identified 22 PsSPLs and conducted a genome-wide analysis of their physical characteristics, chromosome distribution, gene structure, phylogenetic evolution and gene expression patterns. As a result, the PsSPLs were unevenly distributed on the seven chromosomes of pea and harbored the SBP domain, which is composed of approximately 76 amino acid residues. The phylogenetic analysis revealed that the PsSPLs clustered into eight subfamilies and showed high homology with SPL genes in soybean. Further analysis showed the presence of segmental duplications in the PsSPLs. The expression patterns of 22 PsSPLs at different tissues, developmental stages and under various stimulus conditions were evaluated by qRT-PCR method. It was found that the expression patterns of PsSPLs from the same subfamily were similar in different tissues, the transcripts of most PsSPLs reached the maximum peak value at 14 days after anthesis in the pod. Abiotic stresses can cause significantly up-regulated PsSPL19 expression with spatiotemporal specificity, in addition, four plant hormones can cause the up-regulated expression of most PsSPLs including PsSPL19 in a time-dependent manner. Therefore, PsSPL19 could be a key candidate gene for signal transduction during pea growth and development, pod formation, abiotic stress and plant hormone response. Our findings should provide insights for the elucidating of development regulation mechanism and breeding for resistance to abiotic stress pea.

Fingerprint Profiling of Glycans on Extracellular Vesicles via Lectin-Induced Aggregation Strategy for Precise Cancer Diagnostics.

Extracellular vesicles (EVs) harbor abundant glycans that mediate various functions, such as intercellular communication and disease advancement, which play significant roles in disease progression. However, the presence of EV heterogeneity in body fluids and the complex nature of the glycan structures have posed challenges for the detection of EV glycans. In this study, we provide a streamlined method integrated, membrane-specific separation with lectin-induced aggregation strategy (MESSAGE), for multiplexed profiling of EV glycans. By leveraging a rationally designed lectin-induced aggregation strategy, the expression of EV glycans is converted to size-based signals. With the assistance learning machine algorithms, the MESSAGE strategy with high sensitivity, specificity, and simplicity can be used for early cancer diagnosis and classification, as well as monitoring cancer metastasis via 20 µL plasma sample within 2 h. Furthermore, our platform holds promise for advancing the field of EV-based liquid biopsy for clinical applications, opening new possibilities for the profiling of EV glycan signatures in various disease states.

International multicenter study of ultralow graft-to-recipient weight ratio grafts in adult living donor liver transplantation.

Decreasing the graft size in living donor liver transplantation (LDLT) increases the risk of early allograft dysfunction. Graft-to-recipient weight ratio (GRWR) of 0.8 is considered the threshold. There is evidence that smaller volume grafts may also provide equally good outcomes, the cut-off of which remains unknown. In this retrospective multicenter study, 92 adult LDLTs with a final GRWR ≤0.6 performed at 12 international liver transplant centers over a 3-year period were included. Perioperative data including preoperative status, portal flow hemodynamics (PFH) and portal flow modulation, development of small for size syndrome (SFSS), morbidity, and mortality was collated and analyzed. Thirty-two (36.7%) patients developed SFSS and this was associated with increased 30-day, 90-day, and 1-year mortality. The preoperative model for end-stage liver disease and inpatient status were independent predictors for SFSS (P < .05). Pre-liver transplant renal dysfunction was an independent predictor of survival (hazard ratio 3.1; 95% confidence intervals 1.1, 8.9, P = .035). PFH or portal flow modulation were not predictive of SFSS or survival. We report the largest ever multicenter study of LDLT outcomes using ultralow GRWR grafts and for the first time validate the International Liver Transplantation Society-International Living donor liver transplantation study group-Liver Transplantation Society of India consensus definition and grading of SFSS. Preoperative recipient condition rather than GRWR and PFH were independent predictors of SFSS. Algorithms to predict SFSS and LT outcomes should incorporate recipient factors along with GRWR.

Fast Isolation and Sensitive Multicolor Visual Detection of Small Extracellular Vesicles by Multifunctional Polydopamine Nanospheres.

Small extracellular vesicles (sEVs) assume pivotal roles as vital messengers in intercellular communication, boasting a plethora of biological functions and promising clinical applications. However, efficient isolation and sensitive detection of sEVs continue to present formidable challenges. In this study, we report a novel method for fast isolation and highly sensitive multicolor visual detection of sEVs using aptamer-functionalized polydopamine nanospheres (SIMPLE). In the SIMPLE strategy, aptamer-functionalized polydopamine nanospheres (Apt-PDANS) with 170 nm diameters were synthesized and exhibited a remarkable ability to selectively bind to specific proteins on the surface of sEVs. The binding between sEVs and Apt-PDANS engenders an increase in the overall size of the sEVs, allowing fast isolation of sEVs by filtration (a filter membrane with a pore size of 200 nm). The fast isolation strategy not only circumvents the interference posed by unbound proteins and excessive probes as well as the intricacies associated with conventional ultracentrifugation methods but also expedites the separation of sEVs. Concurrently, the incorporation of Fe3+-doped PDANS permits the multicolor visual detection of sEVs, enabling quantitative analysis by the discernment of visual cues. The proposed strategy achieves a detection limit of 3.2 × 104 sEV mL-1 within 1 h, devoid of any reliance on instrumental apparatus. Furthermore, we showcase the potential application of this methodology in epithelial-mesenchymal transition monitoring and cancer diagnosis, while also envisioning its widespread adoption as a straightforward, rapid, sensitive, and versatile platform for disease monitoring and functional exploration.

Well-Paired-Seq2: High-Throughput and High-Sensitivity Strategy for Characterizing Low RNA-Content Cell/Nucleus Transcriptomes.

Single-cell RNA sequencing (scRNA-seq) is a transformative technology that unravels the intricate cellular state heterogeneity. However, the Poisson-dependent cell capture and low sensitivity in scRNA-seq methods pose challenges for throughput and samples with a low RNA-content. Herein, to address these challenges, we present Well-Paired-Seq2 (WPS2), harnessing size-exclusion and quasi-static hydrodynamics for efficient cell capture. WPS2 exploits molecular crowding effect, tailing activity enhancement in reverse transcription, and homogeneous enzymatic reaction in the initial bead-based amplification to achieve 3116 genes and 8447 transcripts with an average of ∼20000 reads per cell. WPS2 detected 1420 more genes and 4864 more transcripts than our previous Well-Paired-Seq. It sensitively characterizes transcriptomes of low RNA-content single cells and nuclei, overcoming the Poisson limit for cell and barcoded bead capture. WPS2 also profiles transcriptomes from frozen clinical samples, revealing heterogeneous tumor copy number variations and intercellular crosstalk in clear cell renal cell carcinomas. Additionally, we provide the first single-cell-level characterization of rare metanephric adenoma (MA) and uncover potential specific markers. With the advantages of high sensitivity and high throughput, WPS2 holds promise for diverse basic and clinical research.

AMAR-seq: Automated Multimodal Sequencing of DNA Methylation, Chromatin Accessibility, and RNA Expression with Single-Cell Resolution.

Parallel single-cell multimodal sequencing is the most intuitive and precise tool for cellular status research. In this study, we propose AMAR-seq to automate methylation, chromatin accessibility, and RNA expression coanalysis with single-cell precision. We validated the accuracy and robustness of AMAR-seq in comparison with standard single-omics methods. The high gene detection rate and genome coverage of AMAR-seq enabled us to establish a genome-wide gene expression regulatory atlas and triple-omics landscape with single base resolution and implement single-cell copy number variation analysis. Applying AMAR-seq to investigate the process of mouse embryonic stem cell differentiation, we revealed the dynamic coupling of the epigenome and transcriptome, which may contribute to unraveling the molecular mechanisms of early embryonic development. Collectively, we propose AMAR-seq for the in-depth and accurate establishment of single-cell multiomics regulatory patterns in a cost-effective, efficient, and automated manner, paving the way for insightful dissection of complex life processes.

Biomass-Derived Materials for Advanced Rechargeable Batteries.

Biomass-derived materials generally exhibit uniform and highly-stable hierarchical porous structures that can hardly be achieved by conventional chemical synthesis and artificial design. When used as electrodes for rechargeable batteries, these structural and compositional advantages often endow the batteries with superior electrochemical performances. This review systematically introduces the innate merits of biomass-derived materials and their applications as the electrode for advanced rechargeable batteries, including lithium-ion batteries, sodium-ion batteries, potassium-ion batteries, and metal-sulfur batteries. In addition, biomass-derived materials as catalyst supports for metal-air batteries, fuel cells, and redox-flow batteries are also included. The major challenges for specific batteries and the strategies for utilizing biomass-derived materials are detailly introduced. Finally, the future development of biomass-derived materials for advanced rechargeable batteries is prospected. This review aims to promote the development of biomass-derived materials in the field of energy storage and provides effective suggestions for building advanced rechargeable batteries.

Metabolic Labeling and Digital Microfluidic Single-Cell Sequencing for Single Bacterial Genotypic-Phenotypic Analysis.

Accurate assessment of phenotypic and genotypic characteristics of bacteria can facilitate comprehensive cataloguing of all the resistance factors for better understanding of antibiotic resistance. However, current methods primarily focus on individual phenotypic or genotypic profiles across different colonies. Here, a Digital microfluidic-based automated assay for whole-genome sequencing of single-antibiotic-resistant bacteria is reported, enabling Genotypic and Phenotypic Analysis of antibiotic-resistant strains (Digital-GPA). Digital-GPA can efficiently isolate and sequence antibiotic-resistant bacteria illuminated by fluorescent D-amino acid (FDAA)-labeling, producing high-quality single-cell amplified genomes (SAGs). This enables identifications of both minor and major mutations, pinpointing substrains with distinctive resistance mechanisms. Digital-GPA can directly process clinical samples to detect and sequence resistant pathogens without bacterial culture, subsequently provide genetic profiles of antibiotic susceptibility, promising to expedite the analysis of hard-to-culture or slow-growing bacteria. Overall, Digital-GPA opens a new avenue for antibiotic resistance analysis by providing accurate and comprehensive molecular profiles of antibiotic resistance at single-cell resolution.

Ultrahigh-Flux Water Nanopumps Generated by Asymmetric Terahertz Absorption.

Controlling active transport of water through membrane channels is essential for advanced nanofluidic devices. Despite advancements in water nanopump design using techniques like short-range invasion and subnanometer-level control, challenges remain facilely and remotely realizing massive waters active transport. Herein, using molecular dynamic simulations, we propose an ultrahigh-flux nanopump, powered by frequency-specific terahertz stimulation, capable of unidirectionally transporting massive water through asymmetric-wettability membrane channels at room temperature without any external pressure. The key physics behind this terahertz-powered water nanopump is revealed to be the energy flow resulting from the asymmetric optical absorption of water.

Differential molecular responses of hemolymph and hepatopancreas of swimming crab, Portunus trituberculatus, infected with Ameson portunus (Microsporidia).

Ameson portunus (Microsporidia) has caused serious economic losses to the aquaculture industry of swimming crab, Portunus trituberculatus. The hemolymph and hepatopancreas are the main immune organs of P. trituberculatus, and the main sites of A. portunus infection. Elucidating the response characteristics of hemolymph and hepatopancreas to microsporidian infection facilitates the development of microsporidiosis prevention and control strategy. This study performed comparative transcriptomic analysis of hemolymph (PTX/PTXA) and hepatopancreas (PTG/PTGA) of P. trituberculatus uninfected and infected with A. portunus. The results showed that there were 223 and 1309 differentially expressed genes (DEGs) in PTX/PTXA and PTG/PTGA, respectively. The lysosome pathway was significantly enriched after the invasion of the hemolymph by A. portunus. Also, immune-related genes were all significantly up-regulated in the hemolymph and hepatopancreas, suggesting that the invasion by A. portunus may activate host immune responses. Unlike hemolymph, antioxidant and detoxification-related genes were also significantly up-regulated in the hepatopancreas. Moreover, metabolism-related genes were significantly down-regulated in the hepatopancreas, suggesting that energy synthesis, resistance to pathogens, and regulation of oxidative stress were suppressed in the hepatopancreas. Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. The differential genes and pathways identified in this study can provide references for the prevention and control of microsporidiosis.

Visible-light-induced decarboxylative cascade cyclization of acryloylbenzamides with N-hydroxyphthalimide esters via EDA complexes.

A visible-light-induced decarboxylative cascade reaction of acryloylbenzamides with alkyl N-hydroxyphthalimide (NHP) esters for the synthesis of various 4-alkyl isoquinolinediones mediated by triphenylphosphine (PPh3) and sodium iodide (NaI) was developed. This operationally simple protocol proceeded via the photoactivation of electron donor-acceptor (EDA) complexes between N-hydroxyphthalimide esters and NaI/PPh3, resulting in multiple carbon-carbon bond formations without the use of precious metal complexes or synthetically elaborate organic dyes, which provided an alternative practical approach to synthesize diverse isoquinoline-1,3(2H,4H)-dione derivatives.

Hepatic arterial infusion therapy for advanced hepatocellular carcinoma after systemic treatment failure: Multicenter, real-world study.

AIM: The study was conducted to evaluate the feasibility and safety profile of hepatic arterial infusion chemotherapy with oxaliplatin, 5-fluorouracil, and leucovorin (HAIC-FOLFOX) as an alternative therapeutic choice for patients with advanced hepatocellular carcinoma (HCC) that is refractory to systemic treatment including immune checkpoint blockades or molecular targeting agents. METHODS: Two hundred and forty five consecutive patients with advanced HCC who received HAIC-FOLFOX treatment after systemic treatment failure were retrospectively reviewed in six institutions and their survival, tumor response, and tolerance were assessed. RESULTS: The median overall survival (OS) and progression-free survival of the 209 included participants were 10.5 months (95% confidence interval [CI], 8.1-12.9) and 6.0 months (95% CI, 5.1-6.9), respectively. According to Response Evaluation Criteria in Solid Tumors 1.1 criteria, the objective response rate was 21.1%, and the disease control rate was 64.6%. Multivariate analysis of risk factors of OS were albumin-bilirubin grade (2 and 3 vs. 1, hazard ratio [HR] 1.57; 95% CI, 1.05-2.34; p = 0.028), tumor number (>3 vs. 1-3, HR 2.18; 95% CI, 1.10-4.34; p = 0.026), extrahepatic spread (present vs. absent, HR 1.61, 95% CI, 1.06-2.45; p = 0.027), synchronous systemic treatment (present vs. absent, HR 0.55, 95% CI, 0.37-0.83; p = 0.004) and treatment response (responder vs. nonresponder, HR 0.30, 95% CI, 0.17-0.53; p < 0.001). Grade 3-4 adverse events (AEs) occurred in 59 (28.2%) HCC patients. All AEs were manageable, and deaths related to hepatic artery infusion chemotherapy treatment were not observed. CONCLUSIONS: Our findings support the effectiveness and safety of HAIC-FOLFOX treatment for patients with advanced HCC who have failed systemic treatment.

Enhanced water permeation through the terahertz-induced phase and diffusion transition in metal-organic framework membranes.

Freshwater scarcity is a pressing global concern, and water desalination has emerged as a promising solution. Metal-organic framework (MOF) membranes have demonstrated exceptional potential in this regard. However, previous efforts to improve the permeability of MOFs have primarily focused on chemical modifications and synthesis rather than exploring physical methods. Using molecular dynamics simulations, we propose that the use of terahertz waves at a specific frequency of 7.5 ± 1.0 THz significantly enhances water permeability across MOF membranes, up to 27-fold, while maintaining effective ion rejection capabilities throughout the process. The mechanism behind this enhancement involves the resonance between the terahertz wave and the hydrogen bond vibrations of water within the MOF. This resonance amplifies the rotational kinetic energy of water molecules, disrupting the hydrogen bonds and causing a phase transition from quasi 1D square ice to a gas-like phase. Additionally, the diffusion behavior shifts from Fickian diffusion to sub-diffusion, resulting in improved water permeation across the MOF membrane. This study highlights the potential of terahertz waves as a physical tool to enhance the permeability of MOFs in water desalination, providing new avenues for efficient water treatment and resource sustainability.

Insights into the differential molecular response of non-germinated and germinated spores of Ameson portunus in vitro by comparative transcriptome analysis.

Ameson portunus, the recently discovered causative agent of "toothpaste disease" of pond-cultured swimming crabs in China has caused enormous economic losses in aquaculture. Understanding the process of spore germination is helpful to elucidate the molecular mechanism of its invasion of host cells. Here, we obtained mature and germinating spores by isolation and purification and in vitro stimulation, respectively. Then, non-germinated and germinated spores were subjected to the comparative transcriptomic analysis to disclose differential molecular responses of these two stages. The highest germination rate, i.e., 71.45 %, was achieved in 0.01 mol/L KOH germination solution. There were 9,609 significantly differentially expressed genes (DEGs), with 685 up-regulated and 8,924 down-regulated DEGs. The up-regulated genes were significantly enriched in ribosome pathway, and the down-regulated genes were significantly enriched in various metabolic pathways, including carbohydrate metabolism, amino acid metabolism and other metabolism. The results suggested that spores require various carbohydrates and amino acids as energy to support their life activities during germination and synthesize large amounts of ribosomal proteins to provide sites for DNA replication, transcription, translation and protein synthesis of the spores of A. portunus within the host cells. Functional genes related to spore germination, such as protein phosphatase CheZ and aquaporin, were also analyzed. The analysis of transcriptome data and identification of functional genes will help to understand the process of spore germination and invasion.

Nonthermal and reversible control of neuronal signaling and behavior by midinfrared stimulation.

Various neuromodulation approaches have been employed to alter neuronal spiking activity and thus regulate brain functions and alleviate neurological disorders. Infrared neural stimulation (INS) could be a potential approach for neuromodulation because it requires no tissue contact and possesses a high spatial resolution. However, the risk of overheating and an unclear mechanism hamper its application. Here we show that midinfrared stimulation (MIRS) with a specific wavelength exerts nonthermal, long-distance, and reversible modulatory effects on ion channel activity, neuronal signaling, and sensorimotor behavior. Patch-clamp recording from mouse neocortical pyramidal cells revealed that MIRS readily provides gain control over spiking activities, inhibiting spiking responses to weak inputs but enhancing those to strong inputs. MIRS also shortens action potential (AP) waveforms by accelerating its repolarization, through an increase in voltage-gated K+ (but not Na+) currents. Molecular dynamics simulations further revealed that MIRS-induced resonance vibration of -C=O bonds at the K+ channel ion selectivity filter contributes to the K+ current increase. Importantly, these effects are readily reversible and independent of temperature increase. At the behavioral level in larval zebrafish, MIRS modulates startle responses by sharply increasing the slope of the sensorimotor input-output curve. Therefore, MIRS represents a promising neuromodulation approach suitable for clinical application.

Genomic anatomy of male-specific microchromosomes in a gynogenetic fish.

Unisexual taxa are commonly considered short-lived as the absence of meiotic recombination is supposed to accumulate deleterious mutations and hinder the creation of genetic diversity. However, the gynogenetic gibel carp (Carassius gibelio) with high genetic diversity and wide ecological distribution has outlived its predicted extinction time of a strict unisexual reproduction population. Unlike other unisexual vertebrates, males associated with supernumerary microchromosomes have been observed in gibel carp, which provides a unique system to explore the rationales underlying male occurrence in unisexual lineage and evolution of unisexual reproduction. Here, we identified a massively expanded satellite DNA cluster on microchromosomes of hexaploid gibel carp via comparing with the ancestral tetraploid crucian carp (Carassius auratus). Based on the satellite cluster, we developed a method for single chromosomal fluorescence microdissection and isolated three male-specific microchromosomes in a male metaphase cell. Genomic anatomy revealed that these male-specific microchromosomes contained homologous sequences of autosomes and abundant repetitive elements. Significantly, several potential male-specific genes with transcriptional activity were identified, among which four and five genes displayed male-specific and male-biased expression in gonads, respectively, during the developmental period of sex determination. Therefore, the male-specific microchromosomes resembling common features of sex chromosomes may be the main driving force for male occurrence in gynogenetic gibel carp, which sheds new light on the evolution of unisexual reproduction.