Hypermethylation of Bmp2 and Fgfr2 Promoter Regions in Bone Marrow Mesenchymal Stem Cells Leads to Bone Loss in Prematurely Aged Mice.

Osteoporosis is an age-related, systemic skeletal disease that poses a significant public health challenge in contemporary society. Development at the epigenetic level is emerging as an important pathogenic mechanism of osteoporosis. Despite indications of a robust association between DNA methylation and osteoporosis development, a comprehensive understanding of the specific role of DNA methylation in osteoporosis remains limited. In this study, significant bone loss was detected at the beginning of eight weeks of age in mouse models of premature aging (SHJHhr mice). We identified a notable upregulation of DNA methyltransferase 3b/3l (Dnmt3b/l) and downregulation of ten eleven translocation dioxygenase 1 (Tet1) in bone marrow mesenchymal stem cells (BMSCs) isolated from SHJHhr mice, along with an increase in the overall 5-methylcytosine (5mC) levels. Moreover, methylation capture sequencing revealed genomic hypermethylation in SHJHhr mice BMSCs. Integrated methylome and transcriptome analyses revealed several crucial methylated genes and networks that are potentially associated with osteoporosis development. Notably, elevated methylation levels of genes linked to the Wnt signaling pathway, particularly bone morphogenetic protein 2 (Bmp2) and fibroblast growth factor receptor (Fgfr2), appeared to compromise the osteogenic differentiation potential of BMSCs. Concurrently, DNA methyltransferase inhibitors attenuated the methylation of the promoter regions of Bmp2 and Fgfr2 and rescued the osteogenic differentiation potential of the BMSCs from SHJHhr mice. In summary, our study provides novel insights into the role of DNA methylation in the development of osteoporosis and suggests promising prospects for employing epigenetic interventions to manage osteoporosis.

Tex46 knockout male mice are sterile secondary to sperm head malformations and failure to penetrate through the zona pellucida.

Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. It is assumed that sperm head shape is important for sperm-zona pellucida (ZP) penetration but research has yet to elucidate why. We generated testis expressed 46 (Tex46) knockout mice to investigate the essential roles of TEX46 in mammalian reproduction. We used RT-PCR to demonstrate that Tex46 was expressed exclusively in the male reproductive tract in mice and humans. We created Tex46-/- mice using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system and analyzed their fertility. Tex46 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis to elucidate relationships between TEX46 and other acrosome biogenesis-related proteins. Mouse and human TEX46 are testis-enriched and encode a transmembrane protein which is conserved from amphibians to mammals. Loss of the mouse TEX46 protein causes male sterility primarily due to abnormal sperm head formation and secondary effects on sperm motility. Tex46 null spermatozoa morphologically lack the typical hooked sperm head appearance and fail to penetrate through the ZP. Electron microscopy of the testicular germ cells reveals malformation of the acrosomal cap, with misshapen sperm head tips and the appearance of a gap between the acrosome head and the nucleus. TEX46 is essential for sperm head formation, sperm penetration through the ZP, and male fertility in mice, and is a putative contraceptive target in men.

Chemical Catalysis Guides Structural Identification for the Major In Vivo Metabolite of the BET Inhibitor JQ1.

The bromodomain inhibitor (+)-JQ1 is a highly validated chemical probe; however, it exhibits poor in vivo pharmacokinetics. To guide efforts toward improving its pharmacological properties, we identified the (+)-JQ1 primary metabolite using chemical catalysis methods. Treatment of (+)-JQ1 with tetrabutylammonium decatungstate under photochemical conditions resulted in selective formation of an aldehyde at the 2-position of the thiophene ring [(+)-JQ1-CHO], which was further reduced to the 2-hydroxymethyl analog [(+)-JQ1-OH]. Comparative LC/MS analysis of (+)-JQ1-OH to the product obtained from liver microsomes suggested (+)-JQ1-OH as the major metabolite of (+)-JQ1. The 2-thienyl position was then substituted to generate a trideuterated (-CD3, (+)-JQ1-D) analog having half-lives that were 1.8- and 2.8-fold longer in mouse and human liver microsomes, respectively. This result unambiguously confirmed (+)-JQ1-OH as the major metabolite of (+)-JQ1. These studies demonstrate an efficient process for studying drug metabolism and identifying the metabolic soft spots of bioactive compounds.

DNA-encoded chemical libraries yield non-covalent and non-peptidic SARS-CoV-2 main protease inhibitors.

The development of SARS-CoV-2 main protease (Mpro) inhibitors for the treatment of COVID-19 has mostly benefitted from X-ray structures and preexisting knowledge of inhibitors; however, an efficient method to generate Mpro inhibitors, which circumvents such information would be advantageous. As an alternative approach, we show here that DNA-encoded chemistry technology (DEC-Tec) can be used to discover inhibitors of Mpro. An affinity selection of a 4-billion-membered DNA-encoded chemical library (DECL) using Mpro as bait produces novel non-covalent and non-peptide-based small molecule inhibitors of Mpro with low nanomolar Ki values. Furthermore, these compounds demonstrate efficacy against mutant forms of Mpro that have shown resistance to the standard-of-care drug nirmatrelvir. Overall, this work demonstrates that DEC-Tec can efficiently generate novel and potent inhibitors without preliminary chemical or structural information.

Osteosarocma progression in biomimetic matrix with different stiffness: Insights from a three-dimensional printed gelatin methacrylamide hydrogel.

Recent studies on osteosarcoma and matrix stiffness are still mostly performed in a 2D setting, which is distinct from in vivo conditions. Therefore, the results from the 2D models may not reflect the real effect of matrix stiffness on cell phenotype. Here, we employed a 3D bioprinted osteosarcoma model, to study the effect of matrix stiffness on osteosarcoma cells. Through density adjustment of GelMA, we constructed three osteosarcoma models with distinct matrix stiffnesses of 50, 80, and 130 kPa. In this study, we found that osteosarcoma cells proliferated faster, migrated more actively, had a more stretched morphology, and a lower drug sensitivity in a softer 3D matrix. When placed in a stiffer matrix, osteosarcoma cells secrete more MMP and VEGF, potentially to fight for survival and attract vascular invasion. Transcriptomic analysis showed that matrix stiffness could impact the signaling pathway of integrin α5-MAPK. The transplantation of 3D printed models in nude mice showed that cells encapsulated in the softer hydrogel were more likely to form subcutaneous tumors. These results suggest that matrix stiffness plays an important role in the development of osteosarcoma in a 3D environment and that inhibition of integrin α5 could block the signal transduction of matrix stiffness.

Constitutively activated AMPKα1 protects against skeletal aging in mice by promoting bone-derived IGF-1 secretion.

Senile osteoporosis is characterized by age-related bone loss and bone microarchitecture deterioration. However, little is known to date about the mechanism that maintains bone homeostasis during aging. In this study, we identify adenosine monophosphate-activated protein kinase alpha 1 (AMPKα1) as a critical factor regulating the senescence and lineage commitment of mesenchymal stem cells (MSCs). A phospho-mutant mouse model shows that constitutive AMPKα1 activation prevents age-related bone loss and promoted MSC osteogenic commitment with increased bone-derived insulin-like growth factor 1 (IGF-1) secretion. Mechanistically, upregulation of IGF-1 signalling by AMPKα1 depends on cAMP-response element binding protein (CREB)-mediated transcriptional regulation. Furthermore, the essential role of the AMPKα1/IGF-1/CREB axis in promoting aged MSC osteogenic potential is confirmed using three-dimensional (3D) culture systems. Taken together, these results can provide mechanistic insight into the protective effect of AMPKα1 against skeletal aging by promoting bone-derived IGF-1 secretion.

ADAD2 functions in spermiogenesis and piRNA biogenesis in mice.

BACKGROUND: Adenosine deaminase domain containing 2 (ADAD2) is a testis-specific protein composed of a double-stranded RNA binding domain and a non-catalytic adenosine deaminase domain. A recent study showed that ADAD2 is indispensable for the male reproduction in mice. However, the detailed functions of ADAD2 remain elusive. OBJECTIVES: This study aimed to investigate the cause of male sterility in Adad2 mutant mice and to understand the molecular functions of ADAD2. MATERIALS AND METHODS: Adad2 homozygous mutant mouse lines, Adad2-/- and Adad2Δ/Δ , were generated by CRISPR/Cas9. Western blotting and immunohistochemistry were used to reveal the expression and subcellular localization of ADAD2. Co-immunoprecipitation tandem mass spectrometry was employed to determine the ADAD2-interacting proteins in mouse testes. RNA-sequencing analyses were carried out to analyze the transcriptome and PIWI-interacting RNA (piRNA) populations in wildtype and Adad2 mutant testes. RESULTS: Adad2-/- and Adad2Δ/Δ mice exhibit male-specific sterility because of abnormal spermiogenesis. ADAD2 interacts with multiple RNA-binding proteins involved in piRNA biogenesis, including MILI, MIWI, RNF17, and YTHDC2. ADAD2 co-localizes and forms novel granules with RNF17 in spermatocytes. Ablation of ADAD2 impairs the formation of RNF17 granules, decreases the number of cluster-derived pachytene piRNAs, and increases expression of ping-pong-derived piRNAs. DISCUSSION AND CONCLUSION: In collaboration with RNF17 and other RNA-binding proteins in spermatocytes, ADAD2 directly or indirectly functions in piRNA biogenesis.

Testis-specific serine kinase 3 is required for sperm morphogenesis and male fertility.

BACKGROUND: The importance of phosphorylation in sperm during spermatogenesis has not been pursued extensively. Testis-specific serine kinase 3 (Tssk3) is a conserved gene, but TSSK3 kinase functions and phosphorylation substrates of TSSK3 are not known. OBJECTIVE: The goals of our studies were to understand the mechanism of action of TSSK3. MATERIALS AND METHODS: We analyzed the localization of TSSK3 in sperm, used CRISPR/Cas9 to generate Tssk3 knockout (KO) mice in which nearly all of the Tssk3 open reading frame was deleted (ensuring it is a null mutation), analyzed the fertility of Tssk3 KO mice by breeding mice for 4 months, and conducted phosphoproteomics analysis of male testicular germ cells. RESULTS: TSSK3 is expressed in elongating sperm and localizes to the sperm tail. To define the essential roles of TSSK3 in vivo, heterozygous (HET) or homozygous KO male mice were mated with wild-type females, and fertility was assessed over 4 months; Tssk3 KO males are sterile, whereas HET males produced normal litter sizes. The absence of TSSK3 results in disorganization of all stages of testicular seminiferous epithelium and significantly increased vacuolization of germ cells, leading to dramatically reduced sperm counts and abnormal sperm morphology; despite these histologic changes, Tssk3 null mice have normal testis size. To elucidate the mechanisms causing the KO phenotype, we conducted phosphoproteomics using purified germ cells from Tssk3 HET and KO testes. We found that proteins implicated in male infertility, such as GAPDHS, ACTL7A, ACTL9, and REEP6, showed significantly reduced phosphorylation in KO testes compared to HET testes, despite unaltered total protein levels. CONCLUSIONS: We demonstrated that TSSK3 is essential for male fertility and crucial for phosphorylation of multiple infertility-related proteins. These studies and the pathways in which TSSK3 functions have implications for human male infertility and nonhormonal contraception.

PDCL2 is essential for sperm acrosome formation and male fertility in mice.

BACKGROUND: Each year, infertility affects 15% of couples worldwide, with 50% of cases attributed to men. Globozoospermia is an uncommon cause of male factor infertility, characterized by defects in sperm acrosome formation, leading to round-headed spermatozoa. OBJECTIVE: We generated Pdcl2 knockout mice to investigate the essential roles of PDCL2 in mammalian reproduction. MATERIALS AND METHODS: We used reverse transcription-polymerase chain reaction to demonstrate that PDCL2 was expressed exclusively in the male reproductive tract in mice and humans. We created Pdcl2 knockout mice using the CRISPR-Cas9 system and analyzed their fertility. Pdcl2 null spermatozoa underwent further evaluation using computer-assisted sperm analysis, light microscopy, and ultrastructural microscopy. We used immunoblot analysis and immunofluorescence to elucidate relationships between PDCL2 and other acrosomal proteins. RESULTS: The PDC family is highly conserved in eukaryotes. Mouse and human PDCL2 are testis enriched and localized to the testicular endoplasmic reticulum. Loss of the protein causes sterility because of abnormal acrosome biogenesis during spermiogenesis and immotility. Furthermore, Pdcl2 null spermatozoa have rounded heads, similar to globozoospermia in humans. Observation of the knockout testis shows a lack of acrosomal cap formation, aberrant localization of mitochondria in the sperm head, and misshapen nuclei. CONCLUSION: PDCL2 is essential for sperm acrosome development and male fertility in mice and is a putative contraceptive target in men.

Advancing ASMS with LC-MS/MS for the discovery of novel PDCL2 ligands from DNA-encoded chemical library selections.

BACKGROUND: A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due to globozoospermia caused by impaired sperm head formation, indicating that PDCL2 is a potential target for male contraception. Herein, our study for the first time developed a biophysical assay for PDCL2 allowing us to screen a series of small molecules, to study structure-activity relationships, and to discover two PDCL2 binders with novel chemical structure. OBJECTIVE: To identify a PDCL2 ligand for therapeutic male contraception, we performed DNA-encoded chemical library (DECL) screening and off-DNA hit validation using a unique affinity selection mass spectrometry (ASMS) biophysical profiling strategy. MATERIALS AND METHODS: We employed the screening process of DECL, which contains billions of chemically unique DNA-barcoded compounds generated through individual sequences of reactions and different combinations of functionalized building blocks. The structures of the PDCL2 binders are proposed based on the sequencing analysis of the DNA barcode attached to each individual DECL compound. The proposed structure is synthesized through multistep reactions. To confirm and determine binding affinity between the DECL identified molecules and PDCL2, we developed an ASMS assay that incorporates liquid chromatography with tandem mass spectrometry (LC-MS/MS). RESULTS: After a screening process of PDCL2 with DECLs containing >440 billion compounds, we identified a series of hits. The selected compounds were synthesized as off-DNA small molecules, characterized by spectroscopy data, and subjected to our ASMS/LC-MS/MS binding assay. By this assay, we discovered two novel compounds, which showed good binding affinity for PDCL2 in comparison to other molecules generated in our laboratory and which were further confirmed by a thermal shift assay. DISCUSSION AND CONCLUSION AND RELEVANCE: With the ASMS/LC-MS/MS assay developed in this paper, we successfully discovered a PDCL2 ligand that warrants further development as a male contraceptive.

A network meta-analysis to evaluate the efficacy of traditional Chinese medicine on intestinal flora in patients with gastrointestinal cancer.

Background and Purpose: Traditional Chinese medicine (TCM) can regulate intestinal flora so as to affect the occurrence, progression, and prognosis of gastrointestinal cancer. According to clinical studies, TCM oral administration, TCM external treatment, and TCM injections, can adjust intestinal flora disorders in patients with gastrointestinal cancer. This network meta-analysis aims to evaluate the effect of three treatments on the intestinal flora in gastrointestinal cancer patients. Methods: This meta-analysis was registered in PROSPERO (CRD42022332553). Six electronic databases, namely CNKI, Wanfang, CSTJ, PubMed, Cochrane Library, and EMBASE, were searched from their inception to 1 April 2022. We identified randomized controlled trials (RCT) used to compare the efficacy of three TCM treatment methods-oral administration, external therapy and injections-on the intestinal flora in gastrointestinal cancer patients. The main outcome indicators were Bifidobacteria, Lactobacilli, Escherichia coli, and Enterococci. Stata (15.1) and the Cochrane risk of bias assessment tool were employed. Results: We identified 20 eligible RCTs with a total of 1,774 patients. According to network meta-analysis results, TCM injection plus common treatment (CT) or oral administration of TCM plus CT was superior to CT alone for supporting Bifidobacterium. In supporting Lactobacillus, TCM injection plus CT demonstrated more obvious effect relative to oral administration of TCM plus CT; TCM injection plus CT was more effective than CT only; and oral administration of TCM plus CT was superior to CT only.The inhibitory effect of TCM injection plus CT on Escherichia coli was better compared with CT only. In terms of inhibiting Enterococci, oral administration of TCM plus CT was superior to CT only.The difference in efficacy among the above treatments was statistically significant. In the SUCRA probability ranking, TCM injection plus CT had the best ranking curve among the three treatments and was the most effective in supporting Bifidobacteria (Sucra = 90.08%), Lactobacilli (Sucra = 96.4%), and regulating Escherichia coli (Sucra = 86.1%) and Enterococci (Sucra = 87.1%). Conclusion: TCM injections plus CT is the most effective therapy in balancing the intestinal flora of gastrointestinal cancer patients. However, the current results deserve further validation through high-quality research. Systematic Review Registration: http://www.prisma-statement.org/, identifier 10.1136/bmj.n71.

Irisin promotes fracture healing by improving osteogenesis and angiogenesis.

Background: Osteogenesis and angiogenesis are important for bone fracture healing. Irisin is a muscle-derived monokine that is associated with bone formation. Methods: To demonstrate the effect of irisin on bone fracture healing, closed mid-diaphyseal femur fractures were produced in 8-week-old C57BL/6 mice. Irisin was administrated intraperitoneally every other day after surgery, fracture healing was assessed by using X-rays. Bone morphometry of the fracture callus were assessed by using micro-computed tomography. Femurs of mice from each group were assessed by the three-point bending testing. Effect of irisin on osteogenic differentiation in mesenchymal stem cells in vitro was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), alkaline phosphatase staining and alizarin red staining. Angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by qRT-PCR, migration tests, and tube formation assays. Results: Increased callus formation, mineralization and tougher fracture healing were observed in the irisin-treated group than in the control group, indicating the better fracture callus healing due to Irisin treatment. The vessel surface and vessel volume fraction of the callus also increased in the irisin-treated group. The expression of BMP2, CD31, and VEGF in callus were enhanced in the irisin-treated group. In mouse bone mesenchymal stem cells, irisin promoted ALP expression and mineralization, and increased the expression of osteogenic genes, including OSX, Runx2, OPG, ALP, OCN and BMP2. Irisin also promoted HUVEC migration and tube formation. Expression of angiogenic genes, including ANGPT1, ANGPT2, VEGFb, CD31, FGF2, and PDGFRB in HUVECs were increased by irisin. Conclusion: All the results indicate irisin can promote fracture healing through osteogenesis and angiogenesis. These findings help in the understanding of muscle-bone interactions during fracture healing. The Translational Potential of this Article: Irisin was one of the most important monokine secreted by skeletal muscle. Studies have found that irisin have anabolic effect one bone remodeling through affecting osteocyte and osteoblast. Based on our study, irisin could promote bone fracture healing by increasing bone mass and vascularization, which provide a potential usage of irisin to promote fracture healing and improve clinical outcomes.

Evaluation of interbody fusion efficacy and biocompatibility of a polyetheretherketone/calcium silicate/porous tantalum cage in a goat model.

Objective: To evaluate the interbody fusion efficacy and biocompatibility of a graft-free cage made of polyetheretherketone/calcium silicate composite/porous tantalum (PEEK/CS/pTa cage) compared with a PEEK/CS cage with an autogenous bone graft in a goat model. Methods: PEEK/CS/pTa and PEEK/CS cages were prepared through an injection-moulding method. The PEEK/CS composites and porous tantalum were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive spectroscopy (EDS) mapping. Then, adult goats were chosen for C2/C3 and C3/C4 discectomy via the anterior cervical approach and randomly implanted with PEEK/CS/pTa and PEEK/CS/cages with autogenous bone grafts. The fusion performance and osseointegration of the cages were evaluated by X-ray imaging, magnetic resonance imaging (MRI) scanning, and bone histomorphometry analysis. Moreover, the concentrations of Ca and Si in urine, serum, tissue around the fusion segments and major organs of the goats were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES). Histological observation of major organs of the goats was used to evaluate the biosafety of PEEK/CS/pTa and PEEK/CS cages. Results: X-ray and MRI imaging suggested that both PEEK/CS/pTa cages and PEEK/CS cages maintained similar average intervertebral space heights. The tissue volumes in the fusion area were comparable between the two groups of cages at 26 weeks after surgery. Histological morphometric data showed that PEEK/CS/pTa cages and PEEK/CS cages with autogenous bone grafts had similar bone contact and osseointegration at 12 and 26 weeks. Element determination of serum, urine, spinal cord, dura matter, bone and organs showed that the CS/PEEK cages did not cause abnormal systemic metabolism or accumulation of calcium and silicon in local tissues and major organs of goats after implantation. No obvious pathological changes were found in the heart, liver, spleen, liver or kidney tissues. Conclusion: Overall, these results suggested that the graft-free PEEK/CS/pTa cage showed similar bony fusion performance to the PEEK/CS cages with autogenous bone grafts. The cages releasing calcium and silicon had good biological safety in vivo.The translational potential of this article: This study provided a new graft-free interbody fusion solution to patients with degenerative disc diseases, which could avert potential donor-site complications. This study also provided a detailed assessment of element excretion and accumulation of Ca and Si in vivo, which validated the biosafety of this new type of bioactive interbody fusion cage.

MRNIP interacts with sex body chromatin to support meiotic progression, spermatogenesis, and male fertility in mice.

Meiosis has a principal role in sexual reproduction to generate haploid gametes in both sexes. During meiosis, the cell nucleus hosts a dynamic environment where some genes are transcriptionally activated, and some are inactivated at the same time. This becomes possible through subnuclear compartmentalization. The sex body, sequestering X and Y chromosomes during male meiosis and creating an environment for the meiotic sex chromosome inactivation (MSCI) is one of the best known and studied subnuclear compartments. Herein, we show that MRNIP forms droplet-like accumulations that fuse together to create a distinct subnuclear compartment that partially overlaps with the sex body chromatin during diplotene. We demonstrate that Mrnip-/- spermatocytes have impaired DNA double-strand break (DSB) repair, they display reduced sex body formation and defective MSCI. We show that Mrnip-/- undergoes critical meiocyte loss at the diplotene stage. Furthermore, we determine that DNA DSBs (induced by SPO11) and synapsis initiation (facilitated by SYCP1) precede Mrnip expression in testes. Altogether, our findings indicate that in addition to an emerging role in DNA DSB repair, MRNIP has an essential function in spermatogenesis during meiosis I by forming drop-like accumulations interacting with the sex body.

The testis-specific E3 ubiquitin ligase RNF133 is required for fecundity in mice.

BACKGROUND: Ubiquitination is a post-translational modification required for a number of physiological functions regulating protein homeostasis, such as protein degradation. The endoplasmic reticulum (ER) quality control system recognizes and degrades proteins no longer needed in the ER through the ubiquitin-proteasome pathway. E2 and E3 enzymes containing a transmembrane domain have been shown to function in ER quality control. The ER transmembrane protein UBE2J1 is a E2 ubiquitin-conjugating enzyme reported to be essential for spermiogenesis at the elongating spermatid stage. Spermatids from Ube2j1 KO male mice are believed to have defects in the dislocation step of ER quality control. However, associated E3 ubiquitin-protein ligases that function during spermatogenesis remain unknown. RESULTS: We identified four evolutionarily conserved testis-specific E3 ubiquitin-protein ligases [RING finger protein 133 (Rnf133); RING finger protein 148 (Rnf148); RING finger protein 151 (Rnf151); and Zinc finger SWIM-type containing 2 (Zswim2)]. Using the CRISPR/Cas9 system, we generated and analyzed the fertility of mutant mice with null alleles for each of these E3-encoding genes, as well as double and triple knockout (KO) mice. Male fertility, male reproductive organ, and sperm-associated parameters were analyzed in detail. Fecundity remained largely unaffected in Rnf148, Rnf151, and Zswim2 KO males; however, Rnf133 KO males displayed severe subfertility. Additionally, Rnf133 KO sperm exhibited abnormal morphology and reduced motility. Ultrastructural analysis demonstrated that cytoplasmic droplets were retained in Rnf133 KO spermatozoa. Although Rnf133 and Rnf148 encode paralogous genes that are chromosomally linked and encode putative ER transmembrane E3 ubiquitin-protein ligases based on their protein structures, there was limited functional redundancy of these proteins. In addition, we identified UBE2J1 as an E2 ubiquitin-conjugating protein that interacts with RNF133. CONCLUSIONS: Our studies reveal that RNF133 is a testis-expressed E3 ubiquitin-protein ligase that plays a critical role for sperm function during spermiogenesis. Based on the presence of a transmembrane domain in RNF133 and its interaction with the ER containing E2 protein UBE2J1, we hypothesize that these ubiquitin-regulatory proteins function together in ER quality control during spermatogenesis.

Comparative Study on Recognition Models of Black-Odorous Water in Hangzhou Based on GF-2 Satellite Data.

To improve the ability of remote sensing technology in recognizing black-odorous water bodies in Hangzhou, this study analyzed the typical spectral characteristics of black-odorous water in Hangzhou based on measured spectral data and water quality parameters, including the transparency, dissolved oxygen, oxidation reduction potential, and ammonia nitrogen. The single-band threshold method, the normalized difference black-odorous water index (NDBWI) model, the black-odorous water index (BOI) model, and the color purity on a Commission Internationale de L'Eclairage (CIE) model were compared to analyze the spatial and temporal distribution characteristics of the black-odorous water in Hangzhou. The results showed that: (1) The remote sensing reflectance of black-odorous water was lower than that of ordinary water, the spectral curve was gentle, and the wave peak shifted toward the near-infrared direction in the wavelength range of 650-850 nm; (2) Among the aforementioned models, the normalized and improved normalized black-odorous water index methods had a higher accuracy, reaching 87.5%, and the threshold values for black-odorous water identification were 0.14 and 0.1, respectively; (3) From 2015 to 2018, the quantity of black-odorous water in the main urban area of Hangzhou showed a decreasing trend, and black-odorous water was mainly distributed in the Gongshu District and tended to appear in narrow rivers, densely populated areas, and factory construction sites. This study is expected to be of great practical value for the rapid tracking and monitoring of urban black-odorous water by using remote sensing technology for future work.

Interfacial Engineering of Binder-Free Janus Separator with Ultra-Thin Multifunctional Layer for Simultaneous Enhancement of Both Metallic Li Anode and Sulfur Cathode.

Exploring a scalable strategy to fabricate a multifunctional separator is of great significance to overcome the challenges of lithium polysulfides (LiPSs) and dendritic growth in lithium-sulfur batteries (LSBs). Herein, a binder-free Janus separator is constructed by interfacial engineering. At the cathode interface, an ultra-thin covalent triazine piperazine film containing tailorable micropores and adsorption sites is decorated on polyacrylonitrile (PAN) membrane by in situ interfacial polymerization, building a triple barrier for LiPSs. The combination of steric hindrance and chemical adsorption reduces LiPS's migration by 81.85%. Meanwhile, at the anode interface, a fast-ionic conductor Li6.4 La3 Zr1.4 Ta0.6 O12  (LLZTO) is created on the surface of PAN nanofiber by magnetron sputtering to suppress dendrite growth. Even though there is no binder between the ceramic layer and the fibrous separator, sputtering creates an inter-embedded structure that ensures no depowering after cycling. Furthermore, the PAN-based separator displays a high temperature tolerance of 180 °C. Consequently, the cell delivers a high capacity of 1287.9 mAh g-1 at 0.5 C and stable cycling performance with an ultra-low capacity decay rate of 0.059% per cycle over 500 cycles. This work provides a scalable strategy for functionalizing separators to tackle the challenges in LSBs, which is binder-free, stripping-free, and essentially thickening-free.

Early Emergent and Progressive Aberrant Subchondral Bone Remodeling Coupled with Aggravated Cartilage Degeneration in Developmental Dysplasia of the Hip.

OBJECTIVE: Developmental dysplasia of the hip (DDH) is the most common skeletal development in children and could result in secondary osteoarthritis. This study aims to clarify the alternations of subchondral trabecular bone remodeling and microstructural properties during the development of DDH, and the potential influence of these alternations on the overlying cartilage degeneration and DDH progression. DESIGN: Traditional straight-leg swaddling method was adopted to establish DDH model in newborn Sprague Dawley rats. Hip joint specimens from normal or DDH rats were used. Typical features of DDH in radiological examination were observed by x-ray analysis. Micro-computed tomography analysis was applied to evaluate the microstructural properties of subchondral bone at postnatal weeks 2, 4, and 6. Histological and immunohistochemical analyses were adopted to appraise subchondral bone remodeling activity and cartilage degeneration. The associations among subchondral bone, articular cartilage, and DDH severity were analyzed via multiple linear regression analysis. RESULTS: Compared with control group, the subchondral bone in DDH group displayed a gradual trend of deteriorated microstructure and worsening biomechanical properties along with aberrant bone remodeling, which might be responsible for the inhibition of stress transmission from the articular cartilage to the subchondral bone and thus leading to the cartilage degeneration and accelerated DDH progression. CONCLUSIONS: Our findings indicate that alternations of subchondral trabecular bone in a time-dependent manner could contribute to the DDH progression and the amelioration on subchondral bone might be a favorable therapeutic candidate for DDH.

Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

Multi-duties for one post: Biodegradable bacterial cellulose-based separator for lithium sulfur batteries.

High-energy density lithium sulfur battery containing highly active materials is more prone to safety hazards. Besides, the infamous shuttle effect of lithium polysulfides (LiPSs) and listless redox kinetic limit its practical applications. Here, a "one-for-all" design concept for separator enabled by interfacial engineering is proposed to relieve the bottlenecks. For one thing, porous bacterial cellulose (PBC) membrane with high thermostability (no shrinking at 200 °C) and puncture resistance was employed to ensure the battery's safety. For another, a difunctional Ti3C2Tx-SnS2 modified layer could capture LiPSs through lewis-acid interaction and promoted the redox kinetics by catalytically active sites. The symmetric cell with anchoring-electrocatalysis Ti3C2Tx-SnS2-PBC separator infiltrated with the electrolyte delivered an ionic conductivity of 2.171 mS/cm at a high temperature of 180 °C. And a capacity retention is improved by 71.2% compared with PP separator. This work furnishes a facial engineering strategy for manufacturing a multifunctional separator for lithium sulfur batteries.