Fast cycling of lithium metal in solid-state batteries by constriction-susceptible anode materials.

Interface reaction between lithium (Li) and materials at the anode is not well understood in an all-solid environment. This paper unveils a new phenomenon of constriction susceptibility for materials at such an interface, the utilization of which helps facilitate the design of an active three-dimensional scaffold to host rapid plating and stripping of a significant amount of a thick Li metal layer. Here we focus on the well-known anode material silicon (Si) to demonstrate that, rather than strong Li-Si alloying at the conventional solid-liquid interface, the lithiation reaction of micrometre-sized Si can be significantly constricted at the solid-solid interface so that it occurs only at thin surface sites of Si particles due to a reaction-induced, diffusion-limiting process. The dynamic interaction between surface lithiation and Li plating of a family of anode materials, as predicted by our constrained ensemble computational approach and represented by Si, silver (Ag) and alloys of magnesium (Mg), can thus more homogeneously distribute current densities for the rapid cycling of Li metal at high areal capacity, which is important in regard to solid-state battery application.

SperMD: the expression atlas of sperm maturation.

The impairment of sperm maturation is one of the major pathogenic factors in male subfertility, a serious medical and social problem affecting millions of global couples. Regrettably, the existing research on sperm maturation is slow, limited, and fragmented, largely attributable to the lack of a global molecular view. To fill the data gap, we newly established a database, namely the Sperm Maturation Database (SperMD, http://bio-add.org/SperMD ). SperMD integrates heterogeneous multi-omics data (170 transcriptomes, 91 proteomes, and five human metabolomes) to illustrate the transcriptional, translational, and metabolic manifestations during the entire lifespan of sperm maturation. These data involve almost all crucial scenarios related to sperm maturation, including the tissue components of the epididymal microenvironment, cell constituents of tissues, different pathological states, and so on. To the best of our knowledge, SperMD could be one of the limited repositories that provide focused and comprehensive information on sperm maturation. Easy-to-use web services are also implemented to enhance the experience of data retrieval and molecular comparison between humans and mice. Furthermore, the manuscript illustrates an example application demonstrated to systematically characterize novel gene functions in sperm maturation. Nevertheless, SperMD undertakes the endeavor to integrate the islanding omics data, offering a panoramic molecular view of how the spermatozoa gain full reproductive abilities. It will serve as a valuable resource for the systematic exploration of sperm maturation and for prioritizing the biomarkers and targets for precise diagnosis and therapy of male subfertility.

ROS-induced oxidative stress is a major contributor to sperm cryoinjury.

STUDY QUESTION: What is the mechanism behind cryoinjury in human sperm, particularly concerning the interplay between reactive oxygen species (ROS) and autophagy, and how does it subsequently affect sperm fate? SUMMARY ANSWER: The freeze-thaw operation induces oxidative stress by generating abundant ROS, which impairs sperm motility and activates autophagy, ultimately guiding the sperm toward programmed cell death such as apoptosis and necrosis, as well as triggering premature capacitation. WHAT IS KNOWN ALREADY: Both ROS-induced oxidative stress and autophagy are thought to exert an influence on the quality of frozen-thawed sperm. STUDY DESIGN, SIZE, DURATION: Overall, 84 semen specimens were collected from young healthy fertile males, with careful quality evaluation. The specimens were split into three groups to investigate the ROS-induced cryoinjury: normal control without any treatment, sperm treated with 0.5 mM hydrogen peroxide (H2O2) for 1 h, and sperm thawed following cryopreservation. Samples from 48 individuals underwent computer-assisted human sperm analysis (CASA) to evaluate sperm quality in response to the treatments. Semen samples from three donors were analyzed for changes in the sperm proteome after H2O2 treatment, and another set of samples from three donors were analyzed for changes following the freeze-thaw process. The other 30 samples were used for fluorescence-staining and western blotting. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm motility parameters, including progressive motility (PR %) and total motility (PR + NP %), were evaluated using the CASA system on a minimum of 200 spermatozoa. The proteomic profiles were determined with label-free mass spectrometry (MS/MS) and protein identification was performed via ion search against the NCBI human database. Subsequently, comprehensive bioinformatics was applied to detect significant proteomic changes and functional enrichment. Fluorescence-staining and western blot analyses were also conducted to confirm the proteomic changes on selected key proteins. The ROS level was measured using 2',7'-dichlorodihydrofluorescein diacetate labeling and the abundance of bioactive mitochondria was determined by evaluating the inner mitochondrial membrane potential (MMP) level. Molecular behaviors of sequestosome-1 (p62 or SQSTM1) and microtubule-associated proteins 1A/1B light chain 3 (LC3) were monitored to evaluate the state of apoptosis in human sperm. Fluorescent probes oxazole yellow (YO-PRO-1) and propidium iodide (PI) were utilized to monitor programmed cell death, namely apoptosis and necrosis. Additionally, gradient concentrations of antioxidant coenzyme Q10 (CoQ10) were introduced to suppress ROS impacts on sperm. MAIN RESULTS AND THE ROLE OF CHANCE: The CASA analysis revealed a significant decrease in sperm motility for both the H2O2-treatment and freeze-thaw groups. Fluorescence staining showed that high ROS levels were produced in the treated sperm and the MMPs were largely reduced. The introduction of CoQ10 at concentrations of 20 and 30 µM resulted in a significant rescue of progressive motility (P < 0.05). The result suggested that excessive ROS could be the major cause of sperm motility impairment, likely by damaging mitochondrial energy generation. Autophagy was significantly activated in sperm when they were under oxidative stress, as evidenced by the upregulation of p62 and the increased conversion of LC3 as well as the upregulation of several autophagy-related proteins, such as charged multivesicular body protein 2a, mitochondrial import receptor subunit TOM22 homolog, and WD repeat domain phosphoinositide-interacting protein 2. Additionally, fluorescent staining indicated the occurrence of apoptosis and necrosis in both H2O2-treated sperm and post-thaw sperm. The cell death process can be suppressed when CoQ10 is introduced, which consolidates the view that ROS could be the major contributor to sperm cryoinjury. The freeze-thaw process could also initiate sperm premature capacitation, demonstrated by the prominent increase in tyrosine phosphorylated proteins, verified with anti-phosphotyrosine antibody and immunofluorescence assays. The upregulation of capacitation-related proteins, such as hyaluronidase 3 and Folate receptor alpha, supported this finding. LARGE SCALE DATA: The data underlying this article are available in the article and its online supplementary material. LIMITATIONS, REASONS FOR CAUTION: The semen samples were obtained exclusively from young, healthy, and fertile males with progressive motility exceeding 60%, which might overemphasize the positive effects while possibly neglecting the negative impacts of cryoinjury. Additionally, the H2O2 treatment conditions in this study may not precisely mimic the oxidative stress experienced by sperm after thawing from cryopreservation, potentially resulting in the omission of certain molecular alterations. WIDER IMPLICATIONS OF THE FINDINGS: This study provides substantial proteomic data for a comprehensive and deeper understanding of the impact of cryopreservation on sperm quality. It will facilitate the design of optimal protocols for utilizing cryopreserved sperm to improve applications, such as ART, and help resolve various adverse situations caused by chemotherapy, radiotherapy, and surgery. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from the Major Innovation Project of Research Institute of National Health Commission (#2022GJZD01-3) and the National Key R&D Program of China (#2018YFC1003600). All authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Structures of the ADGRG2-Gs complex in apo and ligand-bound forms.

Adhesion G protein-coupled receptors are elusive in terms of their structural information and ligands. Here, we solved the cryogenic-electron microscopy (cryo-EM) structure of apo-ADGRG2, an essential membrane receptor for maintaining male fertility, in complex with a Gs trimer. Whereas the formations of two kinks were determinants of the active state, identification of a potential ligand-binding pocket in ADGRG2 facilitated the screening and identification of dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate and deoxycorticosterone as potential ligands of ADGRG2. The cryo-EM structures of DHEA-ADGRG2-Gs provided interaction details for DHEA within the seven transmembrane domains of ADGRG2. Collectively, our data provide a structural basis for the activation and signaling of ADGRG2, as well as characterization of steroid hormones as ADGRG2 ligands, which might be used as useful tools for further functional studies of the orphan ADGRG2.

DNA-encoded chemistry technology yields expedient access to SARS-CoV-2 Mpro inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.

Human adenylate kinase 6 regulates WNK1 (with no lysine kinase-1) phosphorylation states and affects ion homeostasis in NT2 cells.

Adenylate kinase 6 (AK6), a nucleus localized phosphotransferase in mammalians, shows ubiquitously expression and broad substrate activity in different tissues and cell types. Although the function of AK6 has been extensively studied in different cancer cell lines, its role in mammalian germline is still unknown. Here we showed that knockdown of AK6 inhibits cell proliferation and promotes cell apoptosis in human testicular carcinoma (NT2 cells). Co-immunoprecipitation experiment and in vitro pull down assay identified WNK1 (with no lysine kinase-1) as one of the AK6 interacting proteins in NT2 cells. Moreover, we found that AK6 regulates the phosphorylation states of WNK1 (Thr60) and affects phosphorylation level of Akt (Ser473) upon hypotonic condition, probably affecting chloride channel and regulating ion transport and homeostasis in NT2 cells and consequently contributing to the decreased cell proliferation rate. In conclusion, AK6 regulates WNK1 phosphorylation states and affects ion homeostasis in NT2 cells. These findings provide new insights into the function of AK6 and WNK1 in human testicular carcinoma. This work also provides foundation for further mechanism study of AK6 in spermatogenesis.

Rate Dependence on Inductive and Resonance Effects for the Organocatalyzed Enantioselective Conjugate Addition of Alkenyl and Alkynyl Boronic Acids to ß-Indolyl Enones and ß-Pyrrolyl Enones.

Two key factors bear on reaction rates for the conjugate addition of alkenyl boronic acids to heteroaryl-appended enones: the proximity of inductively electron-withdrawing heteroatoms to the site of bond formation and the resonance contribution of available heteroatom lone pairs to stabilize the developing positive charge at the enone ß-position. For the former, the closer the heteroatom is to the enone ß-carbon, the faster the reaction. For the latter, greater resonance stabilization of the benzylic cationic charge accelerates the reaction. Thus, reaction rates are increased by the closer proximity of inductive electron-withdrawing elements, but if resonance effects are involved, then increased rates are observed with electron-donating ability. Evidence for these trends in isomeric substrates is presented, and the application of these insights has allowed for reaction conditions that provide improved reactivity with previously problematic substrates.

Palladium-Catalyzed Hydroxycarbonylation of (Hetero)aryl Halides for DNA-Encoded Chemical Library Synthesis.

A strategy for DNA-compatible, palladium-catalyzed hydroxycarbonylation of (hetero)aryl halides on DNA-chemical conjugates has been developed. This method generally provided the corresponding carboxylic acids in moderate to very good conversions for (hetero)aryl iodides and bromides, and in poor to moderate conversions for (hetero)aryl chlorides. These conditions were further validated by application within a DNA-encoded chemical library synthesis and subsequent discovery of enriched features from the library in selection experiments against two protein targets.

Development of DNA-Compatible Suzuki-Miyaura Reaction in Aqueous Media.

DNA-encoded chemical libraries (DELs) are a cost-effective technology for the discovery of novel chemical probes and drug candidates. A major limiting factor in assembling productive DELs is the availability of DNA-compatible chemical reactions in aqueous media. In an effort to increase the chemical accessibility and structural diversity of small molecules displayed by DELs, we developed a robust Suzuki-Miyaura reaction protocol that is compatible with the DNA structures. By employing a water-soluble Pd-precatalyst, we developed conditions that allow efficient coupling of DNA-linked aryl halides with a wide variety of boronic acids/esters including heteroaryl boronates.

PNIPAm(x)-PPO(36)-PNIPAm(x) thermo-sensitive triblock copolymers: chain conformation and adsorption behavior on a hydrophobic gold surface.

The chain conformations and adsorption behaviors of four thermo-sensitive poly(N-isopropylacrylamide)x-poly(propylene oxide)36-poly(N-isopropylacrylamide)x (PNIPAmx-PPO36-PNIPAmx) triblock copolymers with x values of 15, 33, 75, and 117 in dilute aqueous solutions were investigated by combined techniques of micro-differential scanning calorimetry (micro-DSC), static and dynamic light scattering (SLS & DLS), and the quartz crystal microbalance (QCM). PNIPAm15-PPO36-PNIPAm15 only exhibited the lower critical solution temperature (LCST) of the PPO block, i.e. 25 °C, because the PNIPAm block with x = 15 was too short to maintain its own LCST. With middle lengths x of 33 and 75, the LCSTs of PPO and PNIPAm blocks were observed, respectively. For the longest PNIPAm block with x = 117, only LCST of PNIPAm block dominated, i.e. 32.3 °C. DLS results revealed that the four PNIPAmx-PPO36-PNIPAmx triblock copolymers formed "associate" structures in their dilute aqueous solutions at 20 °C, which was well below the LCSTs of the PPO and PNIPAm blocks. QCM results indicated that the adsorption time constant decreased with increasing adsorption temperature but tended to increase with increasing length x of the PNIPAm block. A complex adsorption behavior with large adsorption amounts was only observed at the corresponding LCST of the PNIPAm block for PNIPAmx-PPO36-PNIPAmx with longer PNIPAm blocks with x = 33, 75, and 117. Furthermore, the adsorbed PNIPAmx-PPO36-PNIPAmx layers obtained at 20 °C were rigid with less energy dissipation.

Structural Understanding for High-Voltage Stabilization of Lithium Cobalt Oxide.

The rapid development of modern consumer electronics is placing higher demands on the lithium cobalt oxide (LiCoO2 ; LCO) cathode that powers them. Increasing operating voltage is exclusively effective in boosting LCO capacity and energy density but is inhibited by the innate high-voltage instability of the LCO structure that serves as the foundation and determinant of its electrochemical behavior in lithium-ion batteries. This has stimulated extensive research on LCO structural stabilization. Here, it is focused on the fundamental structural understanding of LCO cathode from long-term studies. Multi-scale structures concerning LCO bulk and surface and various structural issues along with their origins and corresponding stabilization strategies with specific mechanisms are uncovered and elucidated at length, which will certainly deepen and advance the knowledge of LCO structure and further its inherent relationship with electrochemical performance. Based on these understandings, remaining questions and opportunities for future stabilization of the LCO structure are also emphasized.

Mechanochemically Robust LiCoO2 with Ultrahigh Capacity and Prolonged Cyclability.

Pushing intercalation-type cathode materials to their theoretical capacity often suffers from fragile Li-deficient frameworks and severe lattice strain, leading to mechanical failure issues within the crystal structure and fast capacity fading. This is particularly pronounced in layered oxide cathodes because the intrinsic nature of their structures is susceptible to structural degradation with excessive Li extraction, which remains unsolved yet despite attempts involving elemental doping and surface coating strategies. Herein, a mechanochemical strengthening strategy is developed through a gradient disordering structure to address these challenges and push the LiCoO2 (LCO) layered cathode approaching the capacity limit (256 mAh g-1, up to 93% of Li utilization). This innovative approach also demonstrates exceptional cyclability and rate capability, as validated in practical Ah-level pouch full cells, surpassing the current performance benchmarks. Comprehensive characterizations with multiscale X-ray, electron diffraction, and imaging techniques unveil that the gradient disordering structure notably diminishes the anisotropic lattice strain and exhibits high fatigue resistance, even under extreme delithiation states and harsh operating voltages. Consequently, this designed LCO cathode impedes the growth and propagation of particle cracks, and mitigates irreversible phase transitions. This work sheds light on promising directions toward next-generation high-energy-density battery materials through structural chemistry design.

Reversible male contraception by targeted inhibition of serine/threonine kinase 33.

Men or mice with homozygous serine/threonine kinase 33 (STK33) mutations are sterile owing to defective sperm morphology and motility. To chemically evaluate STK33 for male contraception with STK33-specific inhibitors, we screened our multibillion-compound collection of DNA-encoded chemical libraries, uncovered potent STK33-specific inhibitors, determined the STK33 kinase domain structure bound with a truncated hit CDD-2211, and generated an optimized hit CDD-2807 that demonstrates nanomolar cellular potency (half-maximal inhibitory concentration = 9.2 nanomolar) and favorable metabolic stability. In mice, CDD-2807 exhibited no toxicity, efficiently crossed the blood-testis barrier, did not accumulate in brain, and induced a reversible contraceptive effect that phenocopied genetic STK33 perturbations without altering testis size. Thus, STK33 is a chemically validated, nonhormonal contraceptive target, and CDD-2807 is an effective tool compound.

Mapping of the human testicular proteome and its relationship with that of the epididymis and spermatozoa.

The testis produces male gametes in the germinal epithelium through the development of spermatogonia and spermatocytes into spermatids and immature spermatozoa with the support of Sertoli cells. The flow of spermatozoa into the epididymis is aided by testicular secretions. In the epididymal lumen, spermatozoa and testicular secretions combine with epididymal secretions that promote sperm maturation and storage. We refer to the combined secretions in the epididymis as the sperm-milieu. With two-dimensional-PAGE matrix-assisted laser desorption ionization time-of-flight MS analysis of healthy testes from fertile accident victims, 725 unique proteins were identified from 1920 two-dimensional-gel spots, and a corresponding antibody library was established. This revealed the presence of 240 proteins in the sperm-milieu by Western blotting and the localization of 167 proteins in mature spermatozoa by ICC. These proteins, and those from the epididymal proteome (Li et al. 2010), form the proteomes of the sperm-milieu and the spermatozoa, comprising 525 and 319 proteins, respectively. Individual mapping of the 319 sperm-located proteins to various testicular cell types by immunohistochemistry suggested that 47% were intrinsic sperm proteins (from their presence in spermatids) and 23% were extrinsic sperm proteins, originating from the epididymis and acquired during maturation (from their absence from the germinal epithelium and presence in the epididymal tissue and sperm-milieu). Whereas 408 of 525 proteins in the sperm-milieu proteome were previously identified as abundant epididymal proteins, the remaining 22%, detected by the use of new testicular antibodies, were more likely to be minor proteins common to the testicular proteome, rather than proteins of testicular origin added to spermatozoa during maturation in the epididymis. The characterization of the sperm-milieu proteome and testicular mapping of the sperm-located proteins presented here provide the molecular basis for further studies on the production and maturation of spermatozoa. This could be the basis of development of diagnostic markers and therapeutic targets for infertility or targets for male contraception.

A Hybrid Deep Transfer Learning Model With Kernel Metric for COVID-19 Pneumonia Classification Using Chest CT Images.

Coronavirus disease-2019 (COVID-19) as a new pneumonia which is extremely infectious, the classification of this coronavirus is essential to effectively control the development of the epidemic. Pathological changes in the chest computed tomography (CT) scans are often used as one of the diagnostic criteria of COVID-19. Meanwhile, deep learning-based transfer learning is currently an effective strategy for computer-aided diagnosis (CAD). To further improve the performance of deep transfer learning model used for COVID-19 classification with CT images, in this article, we propose a hybrid model combined with a semi-supervised domain adaption model and extreme learning machine (ELM) classifier, and the application of a novel multikernel correntropy induced loss function in transfer learning is also presented. The proposed model is evaluated on open-source datasets. The experimental results are compared to some baseline models to verify the effectiveness, while adopting accuracy, precision, recall, F1 score and area under curve (AUC) as the evaluation metrics. Experimental results show that the proposed method improves the performance of original model and is more suitable for CT images analysis.

Progress in Research on Key Factors Regulating Lactation Initiation in the Mammary Glands of Dairy Cows.

Lactation initiation refers to a functional change in the mammary organ from a non-lactating state to a lactating state, and a series of cytological changes in the mammary epithelium from a non-secreting state to a secreting state. Like the development of the mammary gland, it is regulated by many factors (including hormones, cytokines, signaling molecules, and proteases). In most non-pregnant animals, a certain degree of lactation also occurs after exposure to specific stimuli, promoting the development of their mammary glands. These specific stimuli can be divided into two categories: before and after parturition. The former inhibits lactation and decreases activity, and the latter promotes lactation and increases activity. Here we present a review of recent progress in research on the key factors of lactation initiation to provide a powerful rationale for the study of the lactation initiation process and mammary gland development.

A Revised Approach to Orthodontic Treatment Monitoring From Oralscan Video.

Research on orthodontic treatment monitoring from oralscan video is a new direction in dental digitalization. We designed an approach to reconstruct, segment, and estimate the pose of individual teeth to measure orthodontic treatment. To handle the semantic gap in heterogeneous data on the condition that they are combined linearly, we present a multimedia interaction network (MIN) to combine heterogeneous information in point cloud segmentation by extending the graph attention mechanism. Moreover, a structure-aware quadruple loss is designed to explore the relation between multiple and diverse unmatched points in point cloud registration. The performance of our approach is evaluated on multiple tooth registration datasets, and extensive experiments show that our approach improves the accuracy by a margin of 1.4% in the inlier ratio on the Aoralscan3 dataset when it is compared with prevailing approaches.

RhoGDIα regulates spermatogenesis through Rac1/cofilin/F-actin signaling.

Spermatogenesis is an extremely complex process, and any obstruction can cause male infertility. RhoGDIα has been identified as a risk of male sterility. In this study, we generate RhoGDIα knockout mice, and find that the males have severely low fertility. The testes from RhoGDIα-/- mice are smaller than that in WT mice. The numbers of spermatogonia and spermatocytes are decreased in RhoGDIα-/- testis. Spermatogenesis is compromised, and spermatocyte meiosis is arrested at zygotene stage in RhoGDIα-/- mice. Acrosome dysplasia is also observed in sperms of the mutant mice. At the molecular level, RhoGDIα deficiency activate the LIMK/cofilin signaling pathway, inhibiting F-actin depolymerization, impairing testis and inducing low fertility in mouse. In addition, the treatment of RhoGDIα-/- mice with Rac1 inhibitor NSC23766 alleviate testis injury and improve sperm quality by inhibiting the LIMK/cofilin/F-actin pathway during spermatogenesis. Together, these findings reveal a previously unrecognized RhoGDIα/Rac1/F-actin-dependent mechanism involved in spermatogenesis and male fertility.

Conditional deletion of Hspa5 leads to spermatogenesis failure and male infertility in mice.

Heat shock proteins (HSPs) have important roles in different developmental stages of spermatogenesis. The heat shock 70 kDa protein 5 (HSPA5) is an important component of the unfolded protein response that promotes cell survival under endoplasmic reticulum (ER) stress conditions. In this study, we explored the function of HSPA5 in spermatogenesis, by generating a germ cell-specific deletion mutant of the Hspa5 gene (conditional knockout of the Hspa5 gene, Hspa5-cKO) using CRISPR/Cas9 technology and the Cre/Loxp system. Hspa5 knockout resulted in severe germ cell loss and vacuolar degeneration of seminiferous tubules, leading to complete arrest of spermatogenesis, testicular atrophy, and male infertility in adult mice. Furthermore, defects occurred in the spermatogenic epithelium of Hspa5-cKO mice as early as Cre recombinase expression. Germ cell ablation of Hspa5 impaired spermatogonia proliferation and differentiation from post-natal day 7 (P7) to P10, which led to a dramatic reduction of differentiated spermatogonia, compromised meiosis, and led to impairment of testis development and the disruption of the first wave of spermatogenesis. Consistent with these results, single-cell RNA sequencing (scRNA-seq) analysis showed that germ cells, especially differentiated spermatogonia, were dramatically reduced in Hspa5-cKO testes compared with controls at P10, further confirming that HSPA5 is crucial for germ cell development. These results suggest that HSPA5 is indispensable for normal spermatogenesis and male reproduction in mice.

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.