The deubiquitinase cofactor UAF1 interacts with USP1 and plays an essential role in spermiogenesis.

Spermiogenesis defines the final phase of male germ cell differentiation. While multiple deubiquitinating enzymes have been linked to spermiogenesis, the impacts of deubiquitination on spermiogenesis remain poorly characterized. Here, we investigated the function of UAF1 in mouse spermiogenesis. We selectively deleted Uaf1 in premeiotic germ cells using the Stra8-Cre knock-in mouse strain (Uaf1 sKO), and found that Uaf1 is essential for spermiogenesis and male fertility. Further, UAF1 interacts and colocalizes with USP1 in the testes. Conditional knockout of Uaf1 in testes results in disturbed protein levels and localization of USP1, suggesting that UAF1 regulates spermiogenesis through the function of the deubiquitinating enzyme USP1. Using tandem mass tag-based proteomics, we identified that conditional knockout of Uaf1 in the testes results in reduced levels of proteins that are essential for spermiogenesis. Thus, we conclude that the UAF1/USP1 deubiquitinase complex is essential for normal spermiogenesis by regulating the levels of spermiogenesis-related proteins.

AIM2 regulates autophagy to mitigate oxidative stress in aged mice with acute liver injury.

The cytoplasmic pattern recognition receptor, absent in melanoma 2 (AIM2), detects cytosolic DNA, activating the inflammasome and resulting in pro-inflammatory cytokine production and pyroptotic cell death. Recent research has illuminated AIM2's contributions to PANoptosis and host defense. However, the role of AIM2 in acetaminophen (APAP)-induced hepatoxicity remains enigmatic. In this study, we unveil AIM2's novel function as a negative regulator in the pathogenesis of APAP-induced liver damage in aged mice, independently of inflammasome activation. AIM2-deficient aged mice exhibited heightened lipid accumulation and hepatic triglycerides in comparison to their wild-type counterparts. Strikingly, AIM2 knockout mice subjected to APAP overdose demonstrated intensified liver injury, compromised mitochondrial stability, exacerbated glutathione depletion, diminished autophagy, and elevated levels of phosphorylated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Furthermore, our investigation revealed AIM2's mitochondrial localization; its overexpression in mouse hepatocytes amplified autophagy while dampening JNK phosphorylation. Notably, induction of autophagy through rapamycin administration mitigated serum alanine aminotransferase levels and reduced the necrotic liver area in AIM2-deficient aged mice following APAP overdose. Mechanistically, AIM2 deficiency exacerbated APAP-induced acute liver damage and inflammation in aged mice by intensifying oxidative stress and augmenting the phosphorylation of JNK and ERK. Given its regulatory role in autophagy and lipid peroxidation, AIM2 emerges as a promising therapeutic target for age-related acute liver damage treatment.

TTC6-Mediated Stabilization of the Flagellum Annulus Ensures the Rapid and Directed Motion of Sperm.

Sperm motility and structural integrity are essential for successful fertilization in vivo, and any hindrance of the correct assembly of the axoneme and peri-axonemal structures in the sperm flagellum can lead to fertility problems. While there has been considerable advancement in studying diseases related to the flagellum, the underlying mechanisms that control sperm movement are not yet fully understood. In this study, we reveal that the tetratricopeptide repeat protein 6 (Ttc6) gene, expressed mainly in the testes, plays a crucial role in maintaining male fertility in mice. We further demonstrate that the knockout of Ttc6 in mice results in decreased sperm motility and induces an abnormal circular swimming pattern, consequently leading to male subfertility. Morphological analysis showed an atypical hairpin-like appearance of the spermatozoa, and ultrastructural studies showed unsheathed flagella at the juncture between the midpiece and principal piece. Collectively, these findings suggest that TTC6 plays an essential role in maintaining the stability of the annulus region of the sperm flagellum, thus ensuring the swift and directed motion of sperm.

Large-Scale CRISPR Screen of LDLR Pathogenic Variants.

Familial hypercholesterolemia (FH) is a frequently occurring genetic disorder that is linked to early-onset cardiovascular disease. If left untreated, patients with this condition can develop severe cardiovascular complications. Unfortunately, many patients remain undiagnosed, and even when diagnosed, the treatment is often not optimal. Although mutations in the LDLR gene are the primary cause of FH, predicting whether novel variants are pathogenic is not a straightforward task. Understanding the functionality of LDLR variants is crucial in uncovering the genetic basis of FH. Our study utilized CRISPR/Cas9 cytosine base editors in pooled screens to establish a novel approach for functionally assessing tens of thousands of LDLR variants on a large scale. A total of more than 100 single guide RNAs (sgRNAs) targeting LDLR pathogenic mutations were successfully screened with relatively high accuracy. Out of these, 5 sgRNAs were further subjected to functional verification studies, including 1 in the promoter, 1 in the antisense RNA, 1 in the exon, and 2 in the intron. Except for the variant caused by the sgRNA located at intron 16, the functionalities of the other LDLR variants were all downregulated. The high similarity of LDLR intron sequences may lead to some false positives. Overall, these results confirm the reliability of the large-scale screening strategy for functional analysis of LDLR variants, and the screened candidate pathogenic mutations could be used as an auxiliary means of clinical gene detection to prevent FH-induced heart disease.

RNase H1 facilitates recombinase recruitment by degrading DNA-RNA hybrids during meiosis.

DNA-RNA hybrids play various roles in many physiological progresses, but how this chromatin structure is dynamically regulated during spermatogenesis remains largely unknown. Here, we show that germ cell-specific knockout of Rnaseh1, a specialized enzyme that degrades the RNA within DNA-RNA hybrids, impairs spermatogenesis and causes male infertility. Notably, Rnaseh1 knockout results in incomplete DNA repair and meiotic prophase I arrest. These defects arise from the altered RAD51 and DMC1 recruitment in zygotene spermatocytes. Furthermore, single-molecule experiments show that RNase H1 promotes recombinase recruitment to DNA by degrading RNA within DNA-RNA hybrids and allows nucleoprotein filaments formation. Overall, we uncover a function of RNase H1 in meiotic recombination, during which it processes DNA-RNA hybrids and facilitates recombinase recruitment.

SPIDR is required for homologous recombination during mammalian meiosis.

Meiotic recombinases RAD51 and DMC1 mediate strand exchange in the repair of DNA double-strand breaks (DSBs) by homologous recombination. This is a landmark event of meiosis that ensures genetic diversity in sexually reproducing organisms. However, the regulatory mechanism of DMC1/RAD51-ssDNA nucleoprotein filaments during homologous recombination in mammals has remained largely elusive. Here, we show that SPIDR (scaffold protein involved in DNA repair) regulates the assembly or stability of RAD51/DMC1 on ssDNA. Knockout of Spidr in male mice causes complete meiotic arrest, accompanied by defects in synapsis and crossover formation, which leads to male infertility. In females, loss of Spidr leads to subfertility; some Spidr-/- oocytes are able to complete meiosis. Notably, fertility is rescued partially by ablation of the DNA damage checkpoint kinase CHK2 in Spidr-/- females but not in males. Thus, our study identifies SPIDR as an essential meiotic recombination factor in homologous recombination in mammals.

The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice.

The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles toward nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knockout (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.

Erratum: HMEJ-mediated site-specific integration of a myostatin inhibitor increases skeletal muscle mass in porcine.

[This corrects the article DOI: 10.1016/j.omtn.2021.06.011.].

FLOT1 knockdown inhibits growth of AML cells through triggering apoptosis and pyroptosis.

Acute myeloid leukemia (AML) is a group of hematological malignancies characterized by clonal proliferation of immature myeloid cells. Lipid rafts are highly organized membrane subdomains enriched in cholesterol, sphingolipids, and gangliosides and play roles in regulating apoptosis through subcellular redistribution. Flotillin1 (FLOT1) is a component and also a marker of lipid rafts and had been reported to be involved in the progression of cancers and played important roles in cell death. However, the role of FLOT1 in AML remains to be explored. In this study, we found that increased expression of FLOT1 was correlated with poor clinical outcome in AML patients. Knockdown of FLOT1 in AML cells not only promoted cell death in vitro but also inhibited malignant cells engraftment in vivo. Mechanically, FLOT1 knockdown triggered apoptosis and pyroptosis. FLOT1 overexpression promoted AML cell growth and apoptosis resistance. Our findings indicate that FLOT1 is a prognostic factor of AML and may be a potential target for AML treatment.

Correction: Wang et al. TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis. Biology 2022, 11, 141.

The authors would like to make the following correction to the published paper [...].

Porcine ZC3H11A Is Essential for the Proliferation of Pseudorabies Virus and Porcine Circovirus 2.

Porcine epidemic viruses, such as pseudorabies virus (PRV) and porcine circovirus 2 (PCV2), are among the most economically damaging pathogens affecting the swine industry. Importantly, previous studies have shown that cases of human infection with PRV occur frequently, indicating the considerable risk of PRV transmission from pigs to humans. Zinc finger CCCH-type containing 11A (ZC3H11A) has been confirmed to play a crucial role in maintaining the nuclear export of mRNA under stress in humans, but its role in pigs remains unknown. In this study, we observed that ZC3H11A interacted with the transcription and export complex and played an important role in mRNA export. Specifically, we knocked out ZC3H11A in PK-15 cells with CRISPR/Cas9 and challenged them with PRV and PCV2. The results showed that the proliferation of the virus was significantly inhibited in ZC3H11A-/- cells, indicating that porcine ZC3H11A is indispensable for the proliferation of PRV and PCV2. Furthermore, our study demonstrated that the inactivation of ZC3H11A in host cells also inhibited the proliferation of PRV and PCV2. Taken together, the results of our study indicated that ZC3H11A is important for maintaining the export of mRNAs, which in turn facilitates the proliferation of PRV and PCV2, suggesting that it can be a potential target for producing antiviral pigs and drugs.

TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis.

Human telomerase is a specialized DNA polymerase whose catalytic core includes both TERT and human telomerase RNA (hTR). Telomerase in humans, which is silent in most somatic cells, is activated to maintain the telomere length (TEL) in various types of cancer cells, including melanoma. In the vast majority of tumor cells, the TERT promoter is mutated to promote proliferation and inhibit apoptosis. Here, we exploited NG-ABEmax to revert TERT -146 T to -146 C in melanoma, and successfully obtained TERT promoter revertant mutant cells. These TERT revertant mutant cells exhibited significant growth inhibition both in vitro and in vivo. Moreover, A375-146C/C cells exhibited telomere shortening and the downregulation of TERT at both the transcription and protein levels, and migration and invasion were inhibited. In addition, TERT promoter revertant mutation abrogated the inhibitory effect of mutant TERT on apoptosis via B-cell lymphoma 2 (Bcl-2), ultimately leading to cell death. Collectively, the results of our work demonstrate that reverting mutations in the TERT promoter is a potential therapeutic option for melanoma.

Construction of acid-base bifunctional covalent organic frameworks via Doebner reaction for catalysing cascade reaction.

We report herein a series of quinoline-4-carboxylic acid linked COFs via the multicomponent one-pot in situ Doebner reaction. The obtained acid-base bifunctional COFs are chemically stable and can highly promote one-pot cascade deacetalization-Knoevenagel condensation reaction in a heterogeneous way under solvent-free conditions.

CD5L deficiency attenuate acetaminophen-induced liver damage in mice via regulation of JNK and ERK signaling pathway.

CD5 molecule like (CD5L), a member of the scavenger receptor cysteine-rich domain superfamily, plays a critical role in immune homeostasis and inflammatory disease. Acetaminophen (APAP) is a safe and effective antipyretic analgesic. However, overdose may cause liver damage or even liver failure. APAP hepatotoxicity is characterized by extensive necrotic cell death and a sterile inflammatory response, in which the role of CD5L remains to be investigated. In this study, we found that the expression of CD5L was increased in the livers of mice after APAP overdose. Furthermore, CD5L deficiency reduced the increase of alanine transaminase (ALT) level, histopathologic lesion area, c-Jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK) phosphorylation level, Transferase-Mediated dUTP Nick End-Labeling positive (TUNEL+) cells proportion, vascular endothelial cell permeability and release of inflammatory cytokines induced by excess APAP. Therefore, our findings reveal that CD5L may be a potential therapeutic target for prevention and treatment of APAP-induced liver injury.

HMEJ-mediated site-specific integration of a myostatin inhibitor increases skeletal muscle mass in porcine.

As a robust antagonist of myostatin (MSTN), follistatin (FST) is an important regulator of skeletal muscle development, and the delivery of FST to muscle tissue represents a potential therapeutic strategy for muscular dystrophies. The N terminus and FSI domain of FST are the functional domains for MSTN binding. Here, we aimed to achieve site-specific integration of FSI-I-I, including the signal peptide, N terminus, and three FSI domains, into the last codon of the porcine MSTN gene using a homology-mediated end joining (HMEJ)-based strategy mediated by CRISPR-Cas9. Based on somatic cell nuclear transfer (SCNT) technology, we successfully obtained FSI-I-I knockin pigs. H&E staining of longissimus dorsi and gastrocnemius cross-sections showed larger myofiber sizes in FSI-I-I knockin pigs than in controls. Moreover, the Smad and Erk pathways were inhibited, whereas the PI3k/Akt pathway was activated in FSI-I-I knockin pigs. In addition, the levels of MyoD, Myf5, and MyoG transcription were upregulated while that of MRF4 was downregulated in FSI-I-I knockin pigs. These results indicate that the FSI-I-I gene mediates skeletal muscle hypertrophy through an MSTN-related signaling pathway and the expression of myogenic regulatory factors. Overall, FSI-I-I knockin pigs with hypertrophic muscle tissue hold great promise as a therapeutic model for human muscular dystrophies.

CRISPR/Cas9-Mediated Specific Integration of Fat-1 and IGF-1 at the pRosa26 Locus.

Many researchers have focused on knock-in pigs for site-specific integration, but little attention has been given to genetically modified pigs with the targeted integration of multiple recombinant genes. To establish a multigene targeted knock-in editing system, we used the internal ribosome entry site (IRES) and self-cleaving 2A peptide technology to construct a plasmid coexpressing the fatty acid desaturase (Fat-1) and porcine insulin-like growth factor-1 (IGF-1) genes at equal levels. In this study, pigs were genetically modified with multiple genes that were precisely inserted into the pRosa26 locus by using the clustered regularly spaced short palindrome repeat sequence (CRISPR)/CRISPR-related 9 (Cas9) system and somatic cell nuclear transfer technology (SCNT) in combination. Single copies of the Fat-1 and IGF-1 genes were expressed satisfactorily in various tissues of F0-generation pigs. Importantly, gas chromatography analysis revealed a significantly increased n-3 polyunsaturated fatty acid (PUFA) level in these genetically modified pigs, which led to a significant decrease of the n-6 PUFA/n-3 PUFA ratio from 6.982 to 3.122 (*** p < 0.001). In conclusion, the establishment of an editing system for targeted double-gene knock-in in this study provides a reference for the precise integration of multiple foreign genes and lays a foundation for the development of new transgenic pig breeds with multiple excellent phenotypes.

CD36 deficiency ameliorates drug-induced acute liver injury in mice.

BACKGROUND: Acetaminophen (APAP) overdose causes hepatotoxicity and even acute liver failure. Recent studies indicate that sterile inflammation and innate immune cells may play important roles in damage-induced hepatocytes regeneration and liver repair. The scavenger receptor CD36 has its crucial functions in sterile inflammation. However, the roles of CD36 in APAP induced acute liver injury remain unclear and warrant further investigation. METHODS: WT C57BL/6 J and CD36-/- mice were intraperitoneally injected with APAP (300 mg/kg) after fasting for 16 h. Liver injury was evaluated by serum alanine aminotransferase (ALT) level and liver tissue hematoxylin and eosin (H&E) staining. Liver inflammatory factor expression was determined by real-time polymerase chain reaction (PCR). The protein adducts forming from the metabolite of APAP and the metabolism enzyme cytochrome P450 2E1 (CYP2E1) levels were measured by Western blot. Liver infiltrating macrophages and neutrophils were characterized by flow cytometry. RNA sequencing and Western blot were used to evaluate the effect of damage-associated molecular patterns (DAMP) molecule high mobility group B1 (HMGB1) on WT and CD36-/- macrophages. Moreover, PP2, a Src kinase inhibitor, blocking CD36 signaling, was applied in APAP model. RESULTS: The expression of CD36 was increased in the liver of mice after APAP treatment. Compared with WT mice, APAP treated CD36-/- mice show less liver injury. There was no significant difference in APAP protein adducts and CYP2E1 expression between these two strains. However, reduced pro-inflammatory factor mRNA expression and serum IL-1ß level were observed in APAP treated CD36-/- mice as well as infiltrating macrophages and neutrophils. Moreover, CD36 deficiency impaired the activation of c-Jun N-terminal kinase (JNK) caused by APAP. Interestingly, the lack of CD36 reduced the activation of extracellular regulated protein kinases (Erk) and v-akt murine thymoma viral oncogene homolog (Akt) induced by HMGB1. RNA transcription sequencing data indicated that HMGB1 has a different effect on WT and CD36-/- macrophages. Furthermore, treatment with PP2 attenuated APAP induced mouse liver injury. CONCLUSION: Our data demonstrated that CD36 deficiency ameliorated APAP-induced acute liver injury and inflammatory responses by decreasing JNK activation. CD36 might serve as a new target to reduce acute liver injury.

Paternal USP26 mutations raise Klinefelter syndrome risk in the offspring of mice and humans.

Current understanding holds that Klinefelter syndrome (KS) is not inherited, but arises randomly during meiosis. Whether there is any genetic basis for the origin of KS is unknown. Here, guided by our identification of some USP26 variations apparently associated with KS, we found that knockout of Usp26 in male mice resulted in the production of 41, XXY offspring. USP26 protein is localized at the XY body, and the disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11. The unpaired sex chromosomes then result in XY aneuploid spermatozoa. Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26-mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production. Thus, some KS should originate from XY spermatozoa, and paternal USP26 mutations increase the risk of producing KS offspring.

A new TEX11 mutation causes azoospermia and testicular meiotic arrest.

There are many unknown genetic factors that lead to infertility in nonobstructive azoospermia men. Here, we performed whole-exome sequencing in blood samples obtained from 40 azoospermia patients with meiotic arrest and found a novel c.151_154del (p.D51fs) frame-shift mutation in exon 3 of the testis expressed 11 (TEX11) gene in one patient. Sanger sequencing analysis of the patient and 288 fertile men was performed to validate the mutation. Immunohistochemical analysis showed TEX11 expression in late-pachytene spermatocytes and in round spermatids in fertile human testes. In contrast, testes of the patient with TEX11 mutation underwent meiotic arrest and lacked TEX11 expression. Western blotting of human embryonic kidney (HEK293) cells transfected with a vector for the p.D51fs TEX11 variant detected no TEX11 expression. In conclusion, we identified a novel frame-shift mutation in the TEX11 gene in an azoospermia patient, emphasizing that this gene should be included in genetic screening panels for the clinical evaluation of azoospermia patients.

IGF2 improves the developmental competency and meiotic structure of oocytes from aged mice.

Advanced maternal-age is a major factor adversely affecting oocyte quality, consequently worsening pregnancy outcomes. Thus, developing strategies to reduce the developmental defects associated with advanced maternal-age would benefit older mothers. Multiple growth factors involved in female fertility have been extensively studied; however, the age-related impacts of various growth factors remain poorly studied. In the present study, we identified that levels of insulin-like growth factor 2 (IGF2) are significantly reduced in the serum and oocytes of aged mice. We found that adding IGF2 in culture medium promotes oocyte maturation and significantly increases the proportion of blastocysts: from 41% in the untreated control group to 64% (50 nM IGF2) in aged mice (p < 0.05). Additionally, IGF2 supplementation of the culture medium reduced reactive oxygen species production and the incidence of spindle/chromosome defects. IGF2 increases mitochondrial functional activity in oocytes from aged mice: we detected increased ATP levels, elevated fluorescence intensity of mitochondria, higher mitochondrial membrane potentials, and increased overall protein synthesis, as well as increased autophagy activity and decreased apoptosis. Collectively, our findings demonstrate that IGF2 supplementation in culture media improves oocyte developmental competence and reduces meiotic structure defects in oocytes from aged mice.