Macromolecular condensation buffers intracellular water potential.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions1. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales2,3; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

Tumor circadian clock strength influences metastatic potential and predicts patient prognosis in luminal A breast cancer.

Studies in shift workers and model organisms link circadian disruption to breast cancer. However, molecular circadian rhythms in noncancerous and cancerous human breast tissues and their clinical relevance are largely unknown. We reconstructed rhythms informatically, integrating locally collected, time-stamped biopsies with public datasets. For noncancerous breast tissue, inflammatory, epithelial-mesenchymal transition (EMT), and estrogen responsiveness pathways show circadian modulation. Among tumors, clock correlation analysis demonstrates subtype-specific changes in circadian organization. Luminal A organoids and informatic ordering of luminal A samples exhibit continued, albeit dampened and reprogrammed rhythms. However, CYCLOPS magnitude, a measure of global rhythm strength, varied widely among luminal A samples. Cycling of EMT pathway genes was markedly increased in high-magnitude luminal A tumors. Surprisingly, patients with high-magnitude tumors had reduced 5-y survival. Correspondingly, 3D luminal A cultures show reduced invasion following molecular clock disruption. This study links subtype-specific circadian disruption in breast cancer to EMT, metastatic potential, and prognosis.

Meteorin-like protein/METRNL/Interleukin-41 ameliorates atopic dermatitis-like inflammation.

BACKGROUND: Meteorin-like protein (METRNL)/Interleukin-41 (IL-41) is a novel immune-secreted cytokine/myokine involved in several inflammatory diseases. However, how METRNL exerts its regulatory properties on skin inflammation remains elusive. This study aims to elucidate the functionality and regulatory mechanism of METRNL in atopic dermatitis (AD). METHODS: METRNL levels were determined in skin and serum samples from patients with AD and subsequently verified in the vitamin D3 analogue MC903-induced AD-like mice model. The cellular target of METRNL activity was identified by multiplex immunostaining, single-cell RNA-seq and RNA-seq. RESULTS: METRNL was significantly upregulated in lesions and serum of patients with dermatitis compared to healthy controls (p <.05). Following repeated MC903 exposure, AD model mice displayed elevated levels of METRNL in both ears and serum. Administration of recombinant murine METRNL protein (rmMETRNL) ameliorated allergic skin inflammation and hallmarks of AD in mice, whereas blocking of METRNL signaling led to the opposite. METRNL enhanced ß-Catenin activation, limited the expression of Th2-related molecules that attract the accumulation of Arginase-1 (Arg1)hi macrophages, dendritic cells, and activated mast cells. CONCLUSIONS: METRNL can bind to KIT receptor and subsequently alleviate the allergic inflammation of AD by inhibiting the expansion of immune cells, and downregulating inflammatory gene expression by regulating the level of active WNT pathway molecule ß-Catenin.

Citrulline facilitates the glycolysis, proliferation, and metastasis of lung cancer cells by regulating RAB3C.

Lung cancer (LC) is one of the major malignant diseases threatening human health. The study aimed to identify the effect of citrulline on the malignant phenotype of LC cells and to further disclose the potential molecular mechanism of citrulline in regulating the development of LC, providing a novel molecular biological basis for the clinical treatment of LC. The effects of citrulline on the viability, proliferation, migration, and invasion of LC cells (A549, H1299) were validated by CCK-8, colony formation, EdU, and transwell assays. The cell glycolysis was assessed via determining the glucose uptake, lactate production, ATP levels, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR). RNA-seq and molecular docking were performed to screen for citrulline-binding target proteins. Western blotting experiments were conducted to examine the expression of related signaling pathway molecules. In addition, the impacts of citrulline on LC growth in vivo were investigated by constructing mouse models. Citrulline augmented the viability of LC cells in a concentration and time-dependent manner. The proliferation, migration, invasion, glycolysis, and EMT processes of LC cells were substantially enhanced after citrulline treatment. Bioinformatics analysis indicated that citrulline could bind to RAB3C protein. Western blotting results indicated that citrulline activated the IL-6/STAT3 pathway by binding to RAB3C. In addition, animal experiments disclosed that citrulline promoted tumor growth in mice. Citrulline accelerated the glycolysis and activated the IL6/STAT3 pathway through the RAB3C protein, consequently facilitating the development of LC.

Fast Reaction Kinetics and Commendable Low-Temperature Adaptability of Zinc Batteries Enabled by Aprotic Water-Acetamide Symbiotic Solvation Sheath.

Although rechargeable aqueous zinc batteries are cost effectiveness, intrinsicly safe, and high activity, they are also known for bringing rampant hydrogen evolution reaction and corrosion. While eutectic electrolytes can effectively eliminate these issues, its high viscosity severely reduces the mobility of Zn2+ ions and exhibits poor temperature adaptability. Here, we infuse acetamide molecules with Lewis base and hydrogen bond donors into a solvated shell of Zn[(H2 O)6 ]2+ to create Zn(H2 O)3 (ace)(BF4 )2 . The viscosity of 1ace-1H2 O is 0.032 Pa s, significantly lower than that of 1ace-0H2 O (995.6 Pa s), which improves ionic conductivity (9.56 mS cm-1 ) and shows lower freezing point of -45 °C, as opposed to 1ace-0H2 O of 4.04 mS cm-1 and 12 °C, respectively. The acidity of 1ace-1H2 O is ≈2.8, higher than 0ace-1H2 O at ≈0.76, making side reactions less likely. Furthermore, benefiting from the ZnCO3 /ZnF2 -rich organic/inorganic solid electrolyte interface, the Zn || Zn cells cycle more than 1300 hours at 1 mA cm-2 , and the Zn || Cu operated over 1800 cycles with an average Coulomb efficiency of ≈99.8 %. The Zn || PANI cell cycled over 8500 cycles, with a specific capacity of 99.8 mAh g-1 at 5 A g-1 at room temperature, and operated at -40 °C with a capacity of 66.8 mAh g-1 .

The circadian clock and extracellular matrix homeostasis in aging and age-related diseases.

The extracellular matrix (ECM) is the noncellular scaffolding component present within all tissues and organs. It provides crucial biochemical and biomechanical cues to instruct cellular behavior and has been shown to be under circadian clock regulation, a highly conserved cell-intrinsic timekeeping mechanism that has evolved with the 24-hour rhythmic environment. Aging is a major risk factor for many diseases, including cancer, fibrosis, and neurodegenerative disorders. Both aging and our modern 24/7 society disrupt circadian rhythms, which could contribute to altered ECM homeostasis. Understanding the daily dynamics of ECM and how this mechanism changes with age will have a profound impact on tissue health, disease prevention, and improving treatments. Maintaining rhythmic oscillations has been proposed as a hallmark of health. On the other hand, many hallmarks of aging turn out to be key regulators of circadian timekeeping mechanisms. In this review, we summarize new work linking the ECM with circadian clocks and tissue aging. We discuss how the changes in the biomechanical and biochemical properties of ECM during aging may contribute to circadian clock dysregulation. We also consider how the dampening of clocks with age could compromise the daily dynamic regulation of ECM homeostasis in matrix-rich tissues. This review aims to encourage new concepts and testable hypotheses about the two-way interactions between circadian clocks and ECM in the context of aging.

Tick tock, the cartilage clock.

Osteoarthritis (OA) is the most common age-related joint disease, affecting articular cartilage and other joint structures, causing severe pain and disability. Due to a limited understanding of the underlying disease pathogenesis, there are currently no disease-modifying drugs for OA. Circadian rhythms are generated by cell-intrinsic timekeeping mechanisms which are known to dampen during ageing, increasing disease risks. In this review, we focus on one emerging area of chondrocyte biology, the circadian clocks. We first provide a historical perspective of circadian clock discoveries and the molecular underpinnings. We will then focus on the expression and functions of circadian clocks in articular cartilage, including their rhythmic target genes and pathways, links to ageing, tissue degeneration, and OA, as well as tissue niche-specific entrainment pathways. Further research into cartilage clocks and ageing may have broader implications in the understanding of OA pathogenesis, the standardization of biomarker detection, and the development of novel therapeutic routes for the prevention and management of OA and other musculoskeletal diseases.

Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator.

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.

The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis.

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock "gated" pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock(Δ19) mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.

Influence of the extracellular matrix on cell-intrinsic circadian clocks.

Cell-autonomous circadian clocks coordinate tissue homeostasis with a 24-hourly rhythm. The molecular circadian clock machinery controls tissue- and cell type-specific sets of rhythmic genes. Disruptions of clock mechanisms are linked to an increased risk of acquiring diseases, especially those associated with aging, metabolic dysfunction and cancer. Despite rapid advances in understanding the cyclic outputs of different tissue clocks, less is known about how the clocks adapt to their local niche within tissues. We have discovered that tissue stiffness regulates circadian clocks, and that this occurs in a cell-type-dependent manner. In this Review, we summarise new work linking the extracellular matrix with differential control of circadian clocks. We discuss how the changes in tissue structure and cellular microenvironment that occur throughout life may impact on the molecular control of circadian cycles. We also consider how altered clocks may have downstream impacts on the acquisition of diseases.

Tissue physiology revolving around the clock: circadian rhythms as exemplified by the intervertebral disc.

Circadian clocks in the brain and peripheral tissues temporally coordinate local physiology to align with the 24 hours rhythmic environment through light/darkness, rest/activity and feeding/fasting cycles. Circadian disruptions (during ageing, shift work and jet-lag) have been proposed as a risk factor for degeneration and disease of tissues, including the musculoskeletal system. The intervertebral disc (IVD) in the spine separates the bony vertebrae and permits movement of the spinal column. IVD degeneration is highly prevalent among the ageing population and is a leading cause of lower back pain. The IVD is known to experience diurnal changes in loading patterns driven by the circadian rhythm in rest/activity cycles. In recent years, emerging evidence indicates the existence of molecular circadian clocks within the IVD, disruption to which accelerates tissue ageing and predispose animals to IVD degeneration. The cell-intrinsic circadian clocks in the IVD control key aspects of physiology and pathophysiology by rhythmically regulating the expression of ~3.5% of the IVD transcriptome, allowing cells to cope with the drastic biomechanical and chemical changes that occur throughout the day. Indeed, epidemiological studies on long-term shift workers have shown an increased incidence of lower back pain. In this review, we summarise recent findings of circadian rhythms in health and disease, with the IVD as an exemplar tissue system. We focus on rhythmic IVD functions and discuss implications of utilising biological timing mechanisms to improve tissue health and mitigate degeneration. These findings may have broader implications in chronic rheumatic conditions, given the recent findings of musculoskeletal circadian clocks.

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay.

The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10-2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10-2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.

Epithelial and stromal circadian clocks are inversely regulated by their mechano-matrix environment.

The circadian clock is an autonomous molecular feedback loop inside almost every cell in the body. We have shown that the mammary epithelial circadian clock is regulated by the cellular microenvironment. Moreover, a stiff extracellular matrix dampens the oscillations of the epithelial molecular clock. Here, we extend this analysis to other tissues and cell types, and identify an inverse relationship between circadian clocks in epithelia and fibroblasts. Epithelial cells from mammary gland, lung and skin have significantly stronger oscillations of clock genes in soft 3D microenvironments, compared to stiff 2D environments. Fibroblasts isolated from the same tissues show the opposite response, exhibiting stronger oscillations and more prolonged rhythmicity in stiff microenvironments. RNA analysis identified that a subset of mammary epithelial clock genes, and their regulators, are upregulated in 3D microenvironments in soft compared to stiff gels. Furthermore, the same genes are inversely regulated in fibroblasts isolated from the same tissues. Thus, our data reveal for the first time an intrinsic difference in the regulation of circadian genes in epithelia and fibroblasts.

Preservation of circadian rhythms by the protein folding chaperone, BiP.

Dysregulation of collagen synthesis is associated with disease progression in cancer and fibrosis. Collagen synthesis is coordinated with the circadian clock, which in cancer cells is, curiously, deregulated by endoplasmic reticulum (ER) stress. We hypothesized interplay between circadian rhythm, collagen synthesis, and ER stress in normal cells. Here we show that fibroblasts with ER stress lack circadian rhythms in gene expression upon clock-synchronizing time cues. Overexpression of binding immunoglobulin protein (BiP) or treatment with chemical chaperones strengthens the oscillation amplitude of circadian rhythms. The significance of these findings was explored in tendon, where we showed that BiP expression is ramped preemptively prior to a surge in collagen synthesis at night, thereby preventing protein misfolding and ER stress. In turn, this forestalls activation of the unfolded protein response in order for circadian rhythms to be maintained. Thus, targeting ER stress could be used to modulate circadian rhythm and restore collagen homeostasis in disease.-Pickard, A., Chang, J., Alachkar, N., Calverley, B., Garva, R., Arvan, P., Meng, Q.-J., Kadler, K. E. Preservation of circadian rhythms by the protein folding chaperone, BiP.

Disrupted circadian clocks and altered tissue mechanics in primary human breast tumours.

BACKGROUND: Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer. METHODS: We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy. RESULTS: Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia. CONCLUSION: Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer.

A Functional Comparison of Treatment of Intrinsic Sphincter Deficiency with Muscle-Derived and Adipose Tissue-Derived Stem Cells.

This study investigated the effect of muscle-derived stem cells (MDSCs) and adipose tissue-derived stem cells (ADSCs) in the treatment of stress urinary incontinence (SUI) and their differences in a rat model. MDSCs and ADSC were isolated from rats (n = 10), examined for their properties, and labeled with enhanced green fluorescent protein (EGFP) and ß-galactosidase (ß-gal) gene. Rats received bladder-neck and transurethral sphincter injection of EGFP-labeled MDSCs and ß-gal gene-labeled ADSC and injection of D-Hanks as a control (n = 24 each group). At 0, 15, 30, and 60 days after cells injection, urinary voiding function was assessed by urine dynamics detector. The rats were killed to harvest their urethras for tracking of MDSCs and ADSC. Western blotting and quantitative real-time reverse transcription PCR (qRT-PCR) was performed to detect smooth muscle contents. Urodynamic test showed that MDSCs and ADSC improved the function of urination in rats with intrinsic sphincter deficiency (ISD), and effect of MDSCs-treatment was more pronounced. In addition, histologic analysis showed that the MDSCs and ADSC-treated groups had significantly higher myosin and α-smooth muscle actin (α-SMA) content than the control group. Compared with ADSC-treated groups, the MDSCs-treated groups in myosin and α-SMA content showed the tendency of increase. In summary, MDSCs and ADSCs have obvious effects in the treatment and/or prevention of ISD and transplantation of MDSCs is more effective than ADSC. © 2018 IUBMB Life, 70(10):976-984, 2018.

The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration.

OBJECTIVES: The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. METHODS: Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. RESULTS: Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1ß but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. CONCLUSIONS: We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

Circadian clocks and breast cancer.

Circadian clocks respond to environmental time cues to coordinate 24-hour oscillations in almost every tissue of the body. In the breast, circadian clocks regulate the rhythmic expression of numerous genes. Disrupted expression of circadian genes can alter breast biology and may promote cancer. Here we overview circadian mechanisms, and the connection between the molecular clock and breast biology. We describe how disruption of circadian genes contributes to cancer via multiple mechanisms, and link this to increased tumour risk in women who work irregular shift patterns. Understanding the influence of circadian rhythms on breast cancer could lead to more efficacious therapies, reformed public health policy and improved patient outcome.

Transcription factor ZFP38 is essential for meiosis prophase I in male mice.

The production of haploid gametes by meiosis is a cornerstone of sexual reproduction and maintenance of genome integrity. Zfp38 mRNA is expressed in spermatocytes, indicating that transcription factor ZFP38 has the potential to regulate transcription during meiosis. In this study, we generated Zfp38 conditional knockout mice (Zfp38(flox/flox), Stra8-Cre, hereafter called Zfp38 cKO) and found that spermatogenesis did not progress beyond meiosis prophase I in Zfp38 cKO mice. Using a chromosomal spread technique, we observed that Zfp38 cKO spermatocytes exhibited a failure in chromosomal synapsis observed by SYCP1/SYCP3 double staining. Progression of DNA double-strand breaks (DSB) repair is disrupted in Zfp38 cKO spermatocytes, as revealed by γ-H2AX, RAD51 and MLH1 staining. Furthermore, the mRNA and protein levels of DSB repair enzymes and factors that guide their loading onto sites of DSBs, such as RAD51, DMC1, RAD51, TEX15 and PALB2, were significantly reduced in Zfp38 cKO spermatocytes. Taken together, our data suggest that ZFP38 is critical for the chromosomal synapsis and DSB repairs partially via its regulation of DSB repair-associated protein expression during meiotic progression in mouse.