Phospholipid Type Regulates Protein Corona Composition and In Vivo Performance of Lipid Nanodiscs.

Over the years, there has been significant interest in PEGylated lipid-based nanocarriers within the drug delivery field. The inevitable interplay between the nanocarriers and plasma protein plays a pivotal role in their in vivo biological fate. Understanding the factors influencing lipid-based nanocarrier and protein corona interactions is of paramount importance in the design and clinical translation of these nanocarriers. Herein, discoid-shaped lipid nanodiscs (sNDs) composed of different phospholipids with varied lipid tails and head groups were fabricated. We investigated the impact of phospholipid components on the interaction between sNDs and serum proteins, particle stability, and biodistribution. The results showed that all of these lipid nanodiscs remained stable over a 15 day storage period, while their stability in the blood serum demonstrated significant differences. The sND composed of POPG exhibited the least stability due to its potent complement activation capability, resulting in rapid blood clearance. Furthermore, a negative correlation between the complement activation capability and serum stability was identified. Pharmacokinetic and biodistribution experiments indicated that phospholipid composition did not influence the capability of sNDs to evade the accelerated blood clearance phenomenon. Complement deposition on the sND was inversely associated with the area under the curve. Additionally, all lipid nanodiscs exhibited dominant adsorption of apolipoprotein. Remarkably, the POPC-based lipid nanodisc displayed a significantly higher deposition of apolipoprotein E, contributing to an obvious brain distribution, which provides a promising tool for brain-targeted drug delivery.

Biallelic mutations in ARMC12 cause asthenozoospermia and multiple midpiece defects in humans and mice.

BACKGROUND: Asthenozoospermia is a major factor contributing to male infertility. The mitochondrial sheath (MS), an important organelle in the midpiece of spermatozoa, is crucial to sperm motility. ARMC12 is a mitochondrial peripheral membrane protein. Deletion of Armc12 impairs the arrangement of MS and causes infertility in mice. However, the role of ARMC12 in human asthenozoospermia remains unknown. OBJECTIVE: To study the genetic defects in patients with asthenozoospermia. METHODS: A total of 125 patients with asthenozoospermia and 120 men with proven fertility were recruited. Whole-exome sequencing and Sanger sequencing were performed for genetic analysis. Papanicolaou staining, HE staining, immunofluorescent staining, transmission electron microscopy and field emission scanning electron microscopy were employed to observe the morphological and structural defects of the spermatozoa and testes. Armc12-knockout mice were generated using the CRISPR-Cas9 system. Intracytoplasmic sperm injection was used to treat the patients. RESULTS: Biallelic ARMC12 mutations were identified in three patients, including homozygous mutations in two siblings from a consanguineous family and compound heterozygous mutations in one sporadic patient. ARMC12 is mainly expressed in the midpiece of elongated and late spermatids in the human testis. The patients' spermatozoa displayed multiple midpiece defects, including absent MS and central pair, scattered or forked axoneme and incomplete plasma membrane. Spermatozoa from Armc12-/- mice showed parallel defects in the midpiece. Moreover, two patients were treated with intracytoplasmic sperm injection and achieved good outcomes. CONCLUSION: Our findings prove for the first time that defects in ARMC12 cause asthenozoospermia and multiple midpiece defects in humans.

Safety and Clinical Activity of SHR7390 Monotherapy or Combined With Camrelizumab for Advanced Solid Tumor: Results From Two Phase I Trials.

BACKGROUND: SHR7390 is a novel, selective MEK1/2 inhibitor. Here, we report results from two phase I trials conducted to evaluate the tolerability, safety and antitumor activity of SHR7390 monotherapy for advanced solid tumors and SHR7390 plus camrelizumab for treatment-refractory advanced or metastatic colorectal cancer (CRC). PATIENTS AND METHODS: Patients received SHR7390 alone or combined with fixed-dose camrelizumab (200 mg every 2 weeks) in an accelerated titration scheme to determine the maximum tolerated dose (MTD). A recommended dose for expansion was determined based on the safety and tolerability of the dose-escalation stage. The primary endpoints were dose limiting toxicity (DLT) and MTD. RESULTS: In the SHR7390 monotherapy trial, 16 patients were enrolled. DLTs were reported in the 1.0 mg cohort, and the MTD was 0.75 mg. Grade ≥3 treatment-related adverse events (TRAEs) were recorded in 4 patients (25.0%). No patients achieved objective response. In the SHR7390 combination trial, 22 patients with CRC were enrolled. One DLT was reported in the 0.5 mg cohort and the MTD was not reached. Grade ≥3 TRAEs were observed in 8 patients (36.4%), with the most common being rash (n=4). One grade 5 TRAE (increased intracranial pressure) occurred. Five patients (22.7%) achieved partial response, including one of 3 patients with MSS/MSI-L and BRAF mutant tumors, one of 15 patients with MSS/MSI-L and BRAF wild type tumors, and all 3 patients with MSI-H tumors. CONCLUSIONS: SHR7390 0.5 mg plus camrelizumab showed a manageable safety profile. Preliminary clinical activity was reported regardless of MSI and BRAF status.

Microbial diversity in paddy rhizospheric soils around a large industrial thallium-containing sulfide utilization zone.

Thallium (Tl) is a rare and extremely toxic metal whose toxicity is significantly higher than cadmium (Cd), lead (Pb) and antimony (Sb). The extensive utilization of Tl-bearing minerals, such as mining activities, has led to severe Tl pollution in a variety of natural settings, while little is known to date about its effect on the microbial diversity in paddy soils. Also, the geochemical behavior of Tl in the periodical alterations between dry and wet conditions of paddy soils remains largely unknown. Herein, the sequential extraction method and 16S rRNA gene sequence analysis were adopted to analyze Tl's migration and transformation behavior and the microbial diversity in the paddy soils with different pollution levels. The results indicated that Tl was mainly concentrated in reducible fraction, which is different from other types of soils, and may be closely attributed to the abundance of Fe-Mn (hydr)oxides in the paddy rhizospheric soils. Further analysis revealed that pH, total S, Pb, Sb, Tl and Cd were the dominant environmental factors, and the enrichment level of these potentially toxic metal(loid)s (PTMs) exerted obvious impacts on the diversity and abundance of microorganism in the rhizospheric soils, and regulating microbial community. The geochemical fractionation of Tl was closely correlated to soil microorganisms such as Fe reducing bacteria (Geothrix) and sulfate reducing bacteria (Anaerolinea), playing a critical role in Tl geochemical cycle through redox reaction. Hence, further study on microorganisms of paddy rhizospheric soils is of great significance to the countermeasures for remediating Tl-polluted paddy fields and protect the health of residents.

Ferric citrate-induced colonic mucosal damage associated with oxidative stress, inflammation responses, apoptosis, and the changes of gut microbial composition.

Ferric citrate (FC) has been used as an iron fortifier and nutritional supplement, which is reported to induce colitis in rats, however the underlying mechanism remains to be elucidated. We performed a 16-week study of FC in male healthy C57BL/6 mice (nine-month-old) with oral administration of Ctr (0.9 % NaCl), 1.25 % FC (71 mg/kg/bw), 2.5 % FC (143 mg/kg/bw) and 5 % FC (286 mg/kg/bw). FC-exposure resulted in colon iron accumulation, histological alteration and reduce antioxidant enzyme activities, such as glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC), together with enhanced lipid peroxidation level, including malondialdehyde (MDA) level and 4-Hydroxynonenal (4-HNE) protein expression. Exposure to FC was associated with upregulated levels of the interleukin (IL)- 6, IL-1ß, IL-18, IL-8 and tumor necrosis factor α (TNF-α), while down-regulated levels of IL-4 and IL-10. Exposure to FC was positively associated with the mRNA and protein expressions of cysteine-aspartic proteases (Caspase)- 9, Caspase-3, Bcl-2-associated X protein (Bax), while negatively associated with B-cell lymphoma 2 (Bcl2) in mitochondrial apoptosis signaling pathway. FC-exposure changed the diversity and composition of gut microbes. Additionally, the serum lipopolysaccharide (LPS) contents increased in FC-exposed groups when compared with the control group, while the expression of colonic tight junction proteins (TJPs), such as Claudin-1 and Occludin were decreased. These findings indicate that the colonic mucosal injury induced by FC-exposure are associated with oxidative stress generation, inflammation response and cell apoptosis, as well as the changes in gut microbes diversity and composition.

Folic Acid Enables Targeting Delivery of Lipodiscs by Circumventing IgM-Mediated Opsonization.

Folic acid (FA) is one of the most widely utilized small-molecule ligands for cancer targeted drug delivery. Natural IgM was recently found to avidly absorb on the surface of FA-functionalized liposomes (FA-sLip), negatively regulating the in vivo performance by efficiently activating complement. Herein, FA-functionalized lipodiscs (FA-Disc) were constructed to successfully circumvent IgM-mediated opsonization and retained binding activity with folate receptors in vivo. The FA moiety along with the bound IgM was restricted to the highly curved rim of lipodiscs, leading to IgM incapability of presenting the membrane-bound conformation to trigger complement activation. The C1q docking, C3 binding, and C5a release were blocked and accelerated blood clearance phenomenon was mitigated of FA-Disc. FA-Disc retained folate binding activity and could effectively target folate receptor positive tumors in vivo. The present study provides a useful solution to avoid the negative regulation by IgM and achieve FA-enabled targeting by exploring disc-shaped nanocarriers.

Inhibitors against Two PDZ Domains of MDA-9 Suppressed Migration of Breast Cancer Cells.

Melanoma differentiation-associated gene 9 (MDA-9) is a small adaptor protein with tandem PDZ domains that promotes tumor progression and metastasis in various human cancers. However, it is difficult to develop drug-like small molecules with high affinity due to the narrow groove of the PDZ domains of MDA-9. Herein, we identified four novel hits targeting the PDZ1 and PDZ2 domains of MDA-9, namely PI1A, PI1B, PI2A, and PI2B, using a protein-observed nuclear magnetic resonance (NMR) fragment screening method. We also solved the crystal structure of the MDA-9 PDZ1 domain in complex with PI1B and characterized the binding poses of PDZ1-PI1A and PDZ2-PI2A, guided by transferred paramagnetic relaxation enhancement. The protein-ligand interaction modes were then cross-validated by the mutagenesis of the MDA-9 PDZ domains. Competitive fluorescence polarization experiments demonstrated that PI1A and PI2A blocked the binding of natural substrates to the PDZ1 and PDZ2 domains, respectively. Furthermore, these inhibitors exhibited low cellular toxicity, but suppressed the migration of MDA-MB-231 breast carcinoma cells, which recapitulated the phenotype of MDA-9 knockdown. Our work has paved the way for the development of potent inhibitors using structure-guided fragment ligation in the future.

TRIM67 Implicates in Regulating the Homeostasis and Synaptic Development of Mitral Cells in the Olfactory Bulb.

In recent years, olfactory dysfunction has attracted increasingly more attention as a hallmark symptom of neurodegenerative diseases (ND). Deeply understanding the molecular basis underlying the development of the olfactory bulb (OB) will provide important insights for ND studies and treatments. Now, with a genetic knockout mouse model, we show that TRIM67, a new member of the tripartite motif (TRIM) protein family, plays an important role in regulating the proliferation and development of mitral cells in the OB. TRIM67 is abundantly expressed in the mitral cell layer of the OB. The genetic deletion of TRIM67 in mice leads to excessive proliferation of mitral cells in the OB and defects in its synaptic development, resulting in reduced olfactory function in mice. Finally, we show that TRIM67 may achieve its effect on mitral cells by regulating the Semaphorin 7A/Plexin C1 (Sema7A/PlxnC1) signaling pathway.

Transcriptome analysis reveals the accelerated expression of genes related to photosynthesis and chlorophyll biosynthesis contribution to shade-tolerant in Phoebe bournei.

BACKGROUND: Phoebe bournei (P. bournei) is an important and endemic wood species in China. However, the plantation, nursing, and preservation of P. bournei are often affected by light. To investigate its physiological changes and molecular mechanism of low light tolerance, two-year-old P. bournei seedlings were subjected to different shading conditions. With the increase of light intensity in the shade, the leaf color of P. bournei seedlings became darkened, the aboveground/underground biomass significantly increased, the content of chlorophyll increased and the net photosynthetic rate significantly increased. RESULTS: de novo transcriptome analysis showed that 724 and 3,248 genes were differentially expressed due to low light intensity at T1 (35% light exposure) and T2 (10% light exposure), respectively, when compared to the controls. Furthermore, the differentially expressed genes (DEGs) were implicated in photosynthesis, nitrogen metabolism, plant hormone signal transduction, biosynthesis of secondary metabolites, and protein processing in the endoplasmic reticulum by functional enrichment analysis. Moreover, the expression of HSP, CAB, HEMA1, GSA, DVR, MYB, bHLH, PORA, CAO, GLK, and photosystem I and II complex-related genes significantly increased after low light exposure at T2 and T1. CONCLUSIONS: The present study suggests that the rapid growth of P. bournei seedlings under shading conditions may be the result of the accelerated expression of genes related to photosynthesis and chlorophyll biosynthesis, which enable plants to maintain a high photosynthesis rate even under low light conditions.

Two dimers generated by lithospermic decarboxylation coupling from Danshen.

To further reveal the active ingredients of Danshen, we systematically studied its chemical components and obtained two new lithospermic acid derivatives (compounds 1 and 2) together with five known phenylpropionic acids (compounds 3-7) from the dried rhizomes of Salvia miltiorrhiza. The structures of the two new compounds were determined by multiple spectral analyses (UV, IR, HR-ESI-MS, NMR, and ECD). In addition, the absolute configurations were established by chiral analysis and calculated and experimental circular dichroism spectra. Biological research indicated that compound 1 could significantly inhibit the proliferation of isoproterenol (ISO)-treated cardiac fibroblasts (AC16 cells), and MMP9 was found to be the most likely target of compound 1. The protein expression and mRNA levels of MMP9 were increased in ISO-induced AC16 cells, which could be reversed by treatment with compound 1. Furthermore, this treatment could alleviate the migration and activation of ISO-induced cardiac fibroblasts.

Hepatic NCoR1 deletion exacerbates alcohol-induced liver injury in mice by promoting CCL2-mediated monocyte-derived macrophage infiltration.

Nuclear receptor corepressor 1 (NCoR1) is a corepressor of the epigenetic regulation of gene transcription that has important functions in metabolism and inflammation, but little is known about its role in alcohol-associated liver disease (ALD). In this study, we developed mice with hepatocyte-specific NCoR1 knockout (NCoR1Hep-/-) using the albumin-Cre/LoxP system and investigated the role of NCoR1 in the pathogenesis of ALD and the underlying mechanisms. The traditional alcohol feeding model and NIAAA model of ALD were both established in wild-type and NCoR1Hep-/- mice. We showed that after ALD was established, NCoR1Hep-/- mice had worse liver injury but less steatosis than wild-type mice. We demonstrated that hepatocyte-specific loss of NCoR1 attenuated liver steatosis by promoting fatty acid oxidation by upregulating BMAL1 (a circadian clock component that has been reported to promote peroxisome proliferator activated receptor alpha (PPARα)-mediated fatty ß-oxidation by upregulating de novo lipid synthesis). On the other hand, hepatocyte-specific loss of NCoR1 exacerbated alcohol-induced liver inflammation and oxidative stress by recruiting monocyte-derived macrophages via C-C motif chemokine ligand 2 (CCL2). In the mouse hepatocyte line AML12, NCoR1 knockdown significantly increased ethanol-induced CCL2 release. These results suggest that hepatocyte NCoR1 plays distinct roles in controlling liver inflammation and steatosis, which provides new insights into the development of treatments for steatohepatitis induced by chronic alcohol consumption.

Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma.

HDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%-35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

Evaluating the time of HIV infection, diagnosis and treatment delays based on HIV notification data in Northwest China, 2008­19.

BACKGROUND: Timely diagnosis and treatment are crucial for reducing HIV transmission;therefore, estimating the time from HIV infection to antiretroviral therapy (ART) initiation becomes particularly important for people living with HIV. METHODS: We used a well-characterised CD4 depletion model to estimate the time from HIV infection to initiation of ART and the rate of delayed HIV diagnosis (infection to diagnosis >1year) and treatment initiation (diagnosis to treatment >1year), based on HIV notification data for adults (aged ≥18years) in Xi'an city, China, during 2008-19. RESULTS: Overall, 7402 reported HIV diagnoses were included. We estimated more than two-thirds of HIV infections remained undiagnosed (66.1%, 9489/14 345). The estimated proportion of HIV diagnoses that were delayed (>1year) was 80.3% (5941/7402) during 2008-19, and it increased from 72.7% (32/44) in 2008 to 83.5% (908/1088) in 2019. In contrast, the proportion of cases with delayed treatment (>1year) was 13.1% (971/7402) during 2008-19, and it reduced from 75.0% (33/44) in 2008 to 1.5% (16/1088) in 2019. The estimated median time from HIV infection to diagnosis increased from 5.05 (IQR, 0.27-8.15) years to 5.81 (IQR, 2.31-10.28) years, whereas the time from diagnosis to ART initiation reduced from 3.06 (IQR, 1.01-5.20) years in 2008 to 0.07 (IQR, 0.04-0.12) year in 2019. CONCLUSIONS: Early treatment after diagnosis has significantly improved, but timely diagnosis of HIV infections may still require further improvement. The estimated proportion of undiagnosed HIV cases remains high in 2019 in Xi'an city and is likely to impede effective control.

Diurnal transcriptomics analysis reveals the regulatory role of the circadian rhythm in super-hybrid rice LY2186.

Heterosis, an important biological phenomenon wherein F1 hybrids exhibit better performance than any of their parents, has been widely applied; however, its underlying mechanism remains largely unknown. Here, we studied and compared the dynamic transcriptional profiles of super-hybrid rice LY2186 and its parents at 17 time points during 2 day/night cycles and identified 1552 rhythmic differentially expressed genes (RDGs). Cluster and functional enrichment analyses revealed that the day- and night-phased RDGs were mainly enriched in the photosynthesis and stress response categories, respectively. Regulatory network analysis indicated that circadian-related RDGs are core components in both the day and night phases and extensively regulate downstream genes involved in photosynthesis, starch synthesis, plant hormone signal transduction, and other pathways. Furthermore, among the 282 RDGs mapped onto the quantitative tract loci of small intervals (≤100 genes), 72.3% were significantly enriched in the yield, vigor, and anatomy categories. These findings provide valuable information for exploring heterosis mechanisms further and guiding breeding practices.

Deciphering Protein Corona by scFv-Based Affinity Chromatography.

It remains challenging to precisely decipher the structural and functional characteristics of protein coronas. To overcome the drawbacks frequently occurring in the traditional separation methods, an anti-PEG single-chain variable fragment (PEG-scFv) based affinity chromatography (AfC) was developed to achieve precise and efficient separation of protein coronas on PEGylated liposomes (sLip). His-tagged PEG-scFv could readily capture sLip without affecting protein corona compositions, and separate sLip/protein complex from plasma protein aggregates and endogenous vesicles through the Ni-NTA column. AfC demonstrated 43-fold higher protein corona collecting efficiency than centrifugation, which was extremely crucial for separation of in vivo protein coronas due to the limitation of sample size. AfC evaded contamination by endogenous vesicles and protein aggregates occurring in centrifugation, and reserved the loosely bound proteins, providing an unprecedented approach to deeply decipher protein coronas. The scFv-based AfC also paves new avenues for the separation of protein coronas formed on other nanomedicines.

TRIM67 Deficiency Exacerbates Hypothalamic Inflammation and Fat Accumulation in Obese Mice.

Obesity has achieved the appearance of a global epidemic and is a serious cause for concern. The hypothalamus, as the central regulator of energy homeostasis, plays a critical role in regulating food intake and energy expenditure. In this study, we show that TRIM67 in the hypothalamus was responsive to body-energy homeostasis whilst a deficiency of TRIM67 exacerbated metabolic disorders in high-fat-diet-induced obese mice. We found exacerbated neuroinflammation and apoptosis in the hypothalamus of obese TRIM67 KO mice. We also found reduced BDNF in the hypothalamus, which affected the fat sympathetic nervous system innervation and contributed to lipid accumulation in adipose tissue under high-fat-diet exposure. In this study, we reveal potential implications between TRIM67 and the hypothalamic function responding to energy overuptake as well as a consideration for the therapeutic diagnosis of obesity.

Loss of TRIM67 Attenuates the Progress of Obesity-Induced Non-Alcoholic Fatty Liver Disease.

Obesity is considered as a major cause for the development and progress of non-alcoholic fatty liver disease (NAFLD), which is one of the most prevalent chronic liver diseases worldwide. However, molecular mechanisms that implicate in obesity-driven pathophysiology of NAFLD are not well defined. Here, we report a tripartite motif (TRIM) protein family member-TRIM67-that is hardly expressed in liver but is inducible on obese conditions. Enhanced expression of TRIM67 activates hepatic inflammation to disturb lipid metabolic homeostasis and promote the progress of NAFLD induced by obesity, while the deficiency in TRIM67 is protective against these pathophysiological processes. Finally, we show that the important transcription coactivator PGC-1α implicates in the response of hepatic TRIM67 to obesity.

Ameliorating Effects of TRIM67 against Intestinal Inflammation and Barrier Dysfunction Induced by High Fat Diet in Obese Mice.

Tripartite Motif 67 (TRIM67) is an important member of TRIM family proteins, which participates in different cellular processes including immune response, proliferation, differentiation, carcinogenesis, and apoptosis. In recent years, a high fat diet (HFD) has remained one of the main causes of different metabolic diseases and increases in intestinal permeability as well as inducing intestinal inflammation. The current study investigated the protective effects of TRIM67 in the ileum and colon of obese mice. 4-week-old wild-type (WT) C57BL/6N mice and TRIM67 knockout (KO) C57BL/6N mice were selected and randomly divided into four sub-groups, which were fed with control diet (CTR) or HFD for 14 weeks. Samples were collected at the age of 18 weeks for analysis. To construct an in vitro obesity model, over-expressed IPEC-J2 cells (porcine intestinal cells) with Myc-TRIM67 were stimulated with palmitic acid (PA), and its effects on the expression level of TRM67, inflammatory cytokines, and barrier function were evaluated. The KO mice showed pathological lesions in the ileum and colon and this effect was more obvious in KO mice fed with HFD. In addition, KO mice fed with a HFD or CTR diet had increased intestinal inflammation, intestinal permeability, and oxidative stress compared to that WT mice fed with these diets, respectively. Moreover, IPEC-J2 cells were transfected with TRIM67 plasmid to perform the same experiments after stimulation with PA, and the results were found consistent with the in vivo evaluations. Taken together, our study proved for the first time that HFD and TRIM67 KO mice have synergistic damaging effects on the intestine, while TRIM67 plays an important protective role in HFD-induced intestinal damage.

Biallelic mutations in KATNAL2 cause male infertility due to oligo-astheno-teratozoospermia.

Oligo-astheno-teratozoospermia (OAT) is a common cause of male infertility, and most of idiopathic OAT patients are thought to be caused by genetic defects. Here, we recruited 38 primary infertile patients with the OAT phenotype and 40 adult men with proven fertility for genetic analysis and identified biallelic mutations of KATNAL2 by whole-exome sequencing in two cases. F013/II:1, from a consanguineous family, carried the KATNAL2 c.328C > T:p.Arg110X homozygous mutations. The other carried c.55A > G: p.Lys19Glu and c.169C > T: p Arg57Trp biallelic mutations. None of the KATNAL2 variants were found in the 40 adult men with proven fertility. The spermatozoa from patients with KATNAL2 biallelic mutations exhibited conspicuous defects in maturation, head morphology, and the structure of mitochondrial sheaths and flagella. KATNAL2 was mainly expressed in the pericentriolar material and mitochondrial sheath of the spermatozoa from control subjects, but it was undetectable in the spermatozoa from the patients. Furthermore, Katnal2 null male mice were infertile and displayed an OAT phenotype. Our results proved that the biallelic mutations in KATNAL2 cause male infertility and OAT in humans for the first time, to our knowledge, which could enrich the genetic defect spectrum of OAT and be beneficial for its accurate genetic screening and clinical diagnosis.

Pathogenic variants of ATG4D in infertile men with non-obstructive azoospermia identified using whole-exome sequencing.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility, and it is primarily associated with genetic defects. We performed whole-exome sequencing of 236 patients with NOA and identified a homozygous pathogenic variant of autophagy-related 4D cysteine peptidase (ATG4D) in two siblings from a consanguineous family and compound heterozygous pathogenic variants of ATG4D in two sporadic cases. The expression of LC3B, a regulator of autophagic activity, was significantly decreased, and the apoptosis rate of spermatogenic cells in testicular tissues was increased. Transfection of GC-2spd cells with a ATG4D mutant plasmid (Flag-Atg4dmut ) significantly decreased the expression level of Lc3b and increased the rate of apoptosis. Moreover, a pathogenic variant in X-linked ATG4A and compound heterozygous pathogenic variants of ATG4B were identified in one patient each. All novel variants were segregated by disease phenotype and were predicted to be pathogenic. Our findings revealed that autophagy-related cysteine peptidase family genes may play crucial roles in human spermatogenesis and identified ATG4D as a novel candidate gene for male infertility due to NOA.