Structures of sperm flagellar doublet microtubules expand the genetic spectrum of male infertility.

Sperm motility is crucial for successful fertilization. Highly decorated doublet microtubules (DMTs) form the sperm tail skeleton, which propels the movement of spermatozoa. Using cryo-electron microscopy (cryo-EM) and artificial intelligence (AI)-based modeling, we determined the structures of mouse and human sperm DMTs and built an atomic model of the 48-nm repeat of the mouse sperm DMT. Our analysis revealed 47 DMT-associated proteins, including 45 microtubule inner proteins (MIPs). We identified 10 sperm-specific MIPs, including seven classes of Tektin5 in the lumen of the A tubule and FAM166 family members that bind the intra-tubulin interfaces. Interestingly, the human sperm DMT lacks some MIPs compared with the mouse sperm DMT. We also discovered variants in 10 distinct MIPs associated with a subtype of asthenozoospermia characterized by impaired sperm motility without evident morphological abnormalities. Our study highlights the conservation and tissue/species specificity of DMTs and expands the genetic spectrum of male infertility.

Mechanism of IFT-A polymerization into trains for ciliary transport.

Intraflagellar transport (IFT) is the highly conserved process by which proteins are transported along ciliary microtubules by a train-like polymeric assembly of IFT-A and IFT-B complexes. IFT-A is sandwiched between IFT-B and the ciliary membrane, consistent with its putative role in transporting transmembrane and membrane-associated cargoes. Here, we have used single-particle analysis electron cryomicroscopy (cryo-EM) to determine structures of native IFT-A complexes. We show that subcomplex rearrangements enable IFT-A to polymerize laterally on anterograde IFT trains, revealing a cooperative assembly mechanism. Surprisingly, we discover that binding of IFT-A to IFT-B shields the preferred lipid-binding interface from the ciliary membrane but orients an interconnected network of ß-propeller domains with the capacity to accommodate diverse cargoes toward the ciliary membrane. This work provides a mechanistic basis for understanding IFT-train assembly and cargo interactions.

De novo identification of mammalian ciliary motility proteins using cryo-EM.

Dynein-decorated doublet microtubules (DMTs) are critical components of the oscillatory molecular machine of cilia, the axoneme, and have luminal surfaces patterned periodically by microtubule inner proteins (MIPs). Here we present an atomic model of the 48-nm repeat of a mammalian DMT, derived from a cryoelectron microscopy (cryo-EM) map of the complex isolated from bovine respiratory cilia. The structure uncovers principles of doublet microtubule organization and features specific to vertebrate cilia, including previously unknown MIPs, a luminal bundle of tektin filaments, and a pentameric dynein-docking complex. We identify a mechanism for bridging 48- to 24-nm periodicity across the microtubule wall and show that loss of the proteins involved causes defective ciliary motility and laterality abnormalities in zebrafish and mice. Our structure identifies candidate genes for diagnosis of ciliopathies and provides a framework to understand their functions in driving ciliary motility.

Axonemal structures reveal mechanoregulatory and disease mechanisms.

Motile cilia and flagella beat rhythmically on the surface of cells to power the flow of fluid and to enable spermatozoa and unicellular eukaryotes to swim. In humans, defective ciliary motility can lead to male infertility and a congenital disorder called primary ciliary dyskinesia (PCD), in which impaired clearance of mucus by the cilia causes chronic respiratory infections1. Ciliary movement is generated by the axoneme, a molecular machine consisting of microtubules, ATP-powered dynein motors and regulatory complexes2. The size and complexity of the axoneme has so far prevented the development of an atomic model, hindering efforts to understand how it functions. Here we capitalize on recent developments in artificial intelligence-enabled structure prediction and cryo-electron microscopy (cryo-EM) to determine the structure of the 96-nm modular repeats of axonemes from the flagella of the alga Chlamydomonas reinhardtii and human respiratory cilia. Our atomic models provide insights into the conservation and specialization of axonemes, the interconnectivity between dyneins and their regulators, and the mechanisms that maintain axonemal periodicity. Correlated conformational changes in mechanoregulatory complexes with their associated axonemal dynein motors provide a mechanism for the long-hypothesized mechanotransduction pathway to regulate ciliary motility. Structures of respiratory-cilia doublet microtubules from four individuals with PCD reveal how the loss of individual docking factors can selectively eradicate periodically repeating structures.

SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules.

The cilium-centrosome complex contains triplet, doublet, and singlet microtubules. The lumenal surfaces of each microtubule within this diverse array are decorated by microtubule inner proteins (MIPs). Here, we used single-particle cryo-electron microscopy methods to build atomic models of two types of human ciliary microtubule: the doublet microtubules of multiciliated respiratory cells and the distal singlet microtubules of monoflagellated human spermatozoa. We discover that SPACA9 is a polyspecific MIP capable of binding both microtubule types. SPACA9 forms intralumenal striations in the B tubule of respiratory doublet microtubules and noncontinuous spirals in sperm singlet microtubules. By acquiring new and reanalyzing previous cryo-electron tomography data, we show that SPACA9-like intralumenal striations are common features of different microtubule types in animal cilia. Our structures provide detailed references to help rationalize ciliopathy-causing mutations and position cryo-EM as a tool for the analysis of samples obtained directly from ciliopathy patients.

Structural intermediates in the low pH-induced transition of influenza hemagglutinin.

The hemagglutinin (HA) glycoproteins of influenza viruses play a key role in binding host cell receptors and in mediating virus-host cell membrane fusion during virus infection. Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. While structures of prefusion state and postfusion state of HA have been reported, the intermediate structures remain elusive. Here, we report two distinct low pH intermediate conformations of the influenza virus HA using cryo-electron microscopy (cryo-EM). Our results show that a decrease in pH from 7.8 to 5.2 triggers the release of fusion peptides from the binding pockets and then causes a dramatic conformational change in the central helices, in which the membrane-proximal ends of the central helices unwind to an extended form. Accompanying the conformational changes of the central helices, the stem region of the HA undergoes an anticlockwise rotation of 9.5 degrees and a shift of 15 Å. The HA head, after being stabilized by an antibody, remains unchanged compared to the neutral pH state. Thus, the conformational change of the HA stem region observed in our research is likely to be independent of the HA head. These results provide new insights into the structural transition of HA during virus entry.

The bacteriophage ϕ29 tail possesses a pore-forming loop for cell membrane penetration.

Most bacteriophages are tailed bacteriophages with an isometric or a prolate head attached to a long contractile, long non-contractile, or short non-contractile tail. The tail is a complex machine that plays a central role in host cell recognition and attachment, cell wall and membrane penetration, and viral genome ejection. The mechanisms involved in the penetration of the inner host cell membrane by bacteriophage tails are not well understood. Here we describe structural and functional studies of the bacteriophage ϕ29 tail knob protein gene product 9 (gp9). The 2.0 Šcrystal structure of gp9 shows that six gp9 molecules form a hexameric tube structure with six flexible hydrophobic loops blocking one end of the tube before DNA ejection. Sequence and structural analyses suggest that the loops in the tube could be membrane active. Further biochemical assays and electron microscopy structural analyses show that the six hydrophobic loops in the tube exit upon DNA ejection and form a channel that spans the lipid bilayer of the membrane and allows the release of the bacteriophage genomic DNA, suggesting that cell membrane penetration involves a pore-forming mechanism similar to that of certain non-enveloped eukaryotic viruses. A search of other phage tail proteins identified similar hydrophobic loops, which indicates that a common mechanism might be used for membrane penetration by prokaryotic viruses. These findings suggest that although prokaryotic and eukaryotic viruses use apparently very different mechanisms for infection, they have evolved similar mechanisms for breaching the cell membrane.

Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2.

The trimeric SARS coronavirus (SARS-CoV) surface spike (S) glycoprotein consisting of three S1-S2 heterodimers binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition. Here, we report the structure of the SARS-CoV S glycoprotein in complex with its host cell receptor ACE2 revealed by cryo-electron microscopy (cryo-EM). The complex structure shows that only one receptor-binding domain of the trimeric S glycoprotein binds ACE2 and adopts a protruding "up" conformation. In addition, we studied the structures of the SARS-CoV S glycoprotein and its complexes with ACE2 in different in vitro conditions, which may mimic different conformational states of the S glycoprotein during virus entry. Disassociation of the S1-ACE2 complex from some of the prefusion spikes was observed and characterized. We also characterized the rosette-like structures of the clustered SARS-CoV S2 trimers in the postfusion state observed on electron micrographs. Structural comparisons suggested that the SARS-CoV S glycoprotein retains a prefusion architecture after trypsin cleavage into the S1 and S2 subunits and acidic pH treatment. However, binding to the receptor opens up the receptor-binding domain of S1, which could promote the release of the S1-ACE2 complex and S1 monomers from the prefusion spike and trigger the pre- to postfusion conformational transition.

Early exposure of rotating magnetic fields promotes central nervous regeneration in planarian Girardia sinensis.

Magnetic field exposure is an accepted safe and effective modality for nerve injury. However, it is clinically used only as a supplement or salvage therapy at the later stage of treatment. Here, we used a planarian Girardia sinensis decapitated model to investigate beneficial effects of early rotary non-uniform magnetic fields (RMFs) exposure on central nervous regeneration. Our results clearly indicated that magnetic stimulation induced from early RMFs exposure significantly promoted neural regeneration of planarians. This stimulating effect is frequency and intensity dependent. Optimum effects were obtained when decapitated planarians were cultured at 20 °C, starved for 3 days before head-cutting, and treated with 6 Hz 0.02 T RMFs. At early regeneration stage, RMFs exposure eliminated edema around the wound and facilitated subsequent formation of blastema. It also accelerated cell proliferation and recovery of neuron functionality. Early RMFs exposure up-regulated expression of neural regeneration related proteins, EGR4 and Netrin 2, and mature nerve cell marker proteins, NSE and NPY. These results suggest that RMFs therapy produced early and significant benefit in central nervous regeneration, and should be clinically used at the early stage of neural regeneration, with appropriate optimal frequency and intensity.

The interactions between mitochondria and sarcoplasmic reticulum and the proteome characterization of mitochondrion-associated membrane from rabbit skeletal muscle.

To obtain a comprehensive understanding of proteins involved in mitochondrion-sarcoplasmic reticulum (SR) linking, a catalog of proteins from mitochondrion-associated membrane (MAM) of New Zealand white rabbit skeletal muscle were analyzed by an optimized shotgun proteomic method. The membrane fractions were prepared by differential centrifugation and separated by 1D electrophoresis followed by a highly reproducible, automated LC-MS/MS on the hybrid linear ion trap (LTQ)-Orbitrap mass spectrometer. By integrating as low as 1% false discovery rate as one of the features for quality control method, 459 proteins were identified from both of the two independent MAM preparations. Protein pI value, molecular weight range, and transmembrane region were calculated using bioinformatics softwares. One hundred one proteins were recognized as membrane proteins. This protein database suggested that the MAM preparations composed of proteins from mitochondrion, SR, and transverse-tubule. This result indicated mitochondria physically linked with SR in rabbit skeletal muscle, voltage-dependent anion channel 1 (VDAC1), VDAC2, and VDAC3 might participate in formation of the tethers between SR and mitochondria.

TFE3 fusions direct an oncogenic transcriptional program that drives OXPHOS and unveils vulnerabilities in translocation renal cell carcinoma.

Translocation renal cell carcinoma (tRCC) is an aggressive subtype of kidney cancer driven by TFE3 gene fusions, which act via poorly characterized downstream mechanisms. Here we report that TFE3 fusions transcriptionally rewire tRCCs toward oxidative phosphorylation (OXPHOS), contrasting with the highly glycolytic metabolism of most other renal cancers. This TFE3 fusion-driven OXPHOS program, together with heightened glutathione levels found in renal cancers, renders tRCCs sensitive to reductive stress - a metabolic stress state induced by an imbalance of reducing equivalents. Genome-scale CRISPR screening identifies tRCC-selective vulnerabilities linked to this metabolic state, including EGLN1, which hydroxylates HIF-1α and targets it for proteolysis. Inhibition of EGLN1 compromises tRCC cell growth by stabilizing HIF-1a and promoting metabolic reprogramming away from OXPHOS, thus representing a vulnerability to OXPHOS-dependent tRCC cells. Our study defines a distinctive tRCC-essential metabolic program driven by TFE3 fusions and nominates EGLN1 inhibition as a therapeutic strategy to counteract fusion-induced metabolic rewiring.

Mammalian PIWI-piRNA-target complexes reveal features for broad and efficient target silencing.

The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host-pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis. The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC, consistent with their ability to capture targets more efficiently than EfPiwi piRISC. In the presence of target, the seed gate-which enforces seed-target fidelity in microRNA RISC-adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed-target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA-target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species.

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Ciliary central apparatus structure reveals mechanisms of microtubule patterning.

A pair of extensively modified microtubules form the central apparatus (CA) of the axoneme of most motile cilia, where they regulate ciliary motility. The external surfaces of both CA microtubules are patterned asymmetrically with large protein complexes that repeat every 16 or 32 nm. The composition of these projections and the mechanisms that establish asymmetry and longitudinal periodicity are unknown. Here, by determining cryo-EM structures of the CA microtubules, we identify 48 different CA-associated proteins, which in turn reveal mechanisms for asymmetric and periodic protein binding to microtubules. We identify arc-MIPs, a novel class of microtubule inner protein, that bind laterally across protofilaments and remodel tubulin structure and lattice contacts. The binding mechanisms utilized by CA proteins may be generalizable to other microtubule-associated proteins. These structures establish a foundation to elucidate the contributions of individual CA proteins to ciliary motility and ciliopathies.

The Efficacy and Safety of Zhengqing Fengtongning for Knee Osteoarthritis: A Systematic Review and Meta-Analysis of Randomized Clinical Trials.

BACKGROUND: Zhengqing Fengtongning release tablet (ZQFTN) is a proprietary Chinese medicine preparation of sinomenine, the main active component of the traditional Chinese medicine (TCM) Sinomenium acutum. It is used in China as a complementary and alternative medicine (CAM) for knee osteoarthritis (KOA). The objective of this study was to evaluate the clinical efficacy and safety of ZQFTN in KOA treatment. METHOD: Randomized controlled trials of ZQFTN in KOA treatment were searched in PubMed, Cochrane Library, China National Knowledge Infrastructure, Chinese Scientific Journals Database, and Wanfang database. Two reviewers independently conducted the screening, extracted the data, and assessed the methodological quality. Statistical analysis was performed using RevMan 5.3 software. RESULTS: Eighteen studies were assessed that included 1512 participants (757 in the treatment group and 755 in the control group). The results showed that compared with the control group, the Visual Analogue Scale (standardized mean difference (SMD) = -0.87, 95% confidence interval (CI): [-1.08, -0.66], P < 0.001), Western Ontario and Mc Master University (WOMAC) Osteoarthritis Index pain score (SMD = -0.67, 95% CI: [-0.88, -0.46], P < 0.001), WOMAC stiffness score (SMD = -0.53, 95% CI: [-0.86, -0.20], P=0.001), WOMAC function score (SMD = -0.76, 95% CI: [-0.97, -0.55], P < 0.001), serum interleukin-1ß level (SMD = -4.36, 95% CI: [-6.41, -2.31], P < 0.001), and serum tumor necrosis factor-α level (SMD = -8.45, 95% CI: [-11.20, -5.69], P < 0.001) of the ZQFTN treatment group were lower, and the total effective rate was higher relative risk (RR = 1.15, 95% CI [1.07, 1.23], P < 0.001). There was no significant difference in the incidence of adverse reactions between the two groups (RR = 0.96, 95% CI: [0.69, 1.35], P=0.82). CONCLUSION: ZQFTN can effectively relieve knee pain, morning stiffness, and daily activity function disorders, reduce the expression of inflammatory factors in serum, and improve the total clinical response rate without increasing the incidence of adverse reactions. Therefore, ZQFTN has considerable potential as a CAM for KOA. However, due to the limitation of the quality of the included studies, the strength of this conclusion is affected. In the next step, multicenter, large sample, high-quality randomized controlled studies are needed to further confirm the present conclusion.

Sinomenine hydrochloride injection for knee osteoarthritis: A protocol for systematic review and meta-analysis.

BACKGROUND: Knee osteoarthritis (KOA) is a degenerative disease in the knee joint, with chronic joint pain, swelling, stiffness, and dysfunction as the primary manifestations. Sinomenine hydrochloride injection is a proprietary Chinese medicine injection of sinomenine, the main active component of traditional Chinese medicine (TCM). Clinical studies show that Sinomenine hydrochloride injection has a good effect on the treatment of KOA. At present, there is still a lack of systematic reviews and meta-analyses to evaluate the efficacy and safety of sinomenine hydrochloride injection in the treatment of KOA. Our purpose is to supplement this deficiency. METHODS: Randomized controlled trials of sinomenine hydrochloride injection in the treatment of KOA were searched for Eight electronic resource databases. We will use Review Manager 5.3 software for heterogeneity assessment, meta-analysis, and subgroup analysis. We will use the Cochrane Manual to assess the quality of the included studies, and use reporting biases assessment and sensitivity analysis to evaluate the reliability and stability of the results. RESULTS: This study will provide a high-quality synthesis to assess the efficacy and safety of sinomenine hydrochloride injection in the treatment of KOA. CONCLUSION: This systematic review evaluates the efficacy and safety of sinomenine hydrochloride injection in the treatment of KOA. INPLASY REGISTRATION NUMBER: INPLASY2021110057.

[N'-(3-Meth-oxy-2-oxidobenzyl-idene-κO)benzohydrazidato-κN',O]tris-(pyridine-κN)cobalt(III) perchlorate.

In the mononuclear title compound, [Co(III)(C(15)H(12)N(2)O(3))(C(5)H(5)N)(3)]ClO(4), the Co(III) ion is coordinated by three pyridine mol-ecules and one N'-(3-meth-oxy-2-oxidobenzyl-idene)benzohydrazidate Schiff base ligand in an O,N,O'-tridentate manner. The Co(III) ion adopts a distorted CoN(4)O(2) octa-hedral coordination environment.

Structures of radial spokes and associated complexes important for ciliary motility.

In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii. We determine structures of isolated radial spoke RS1 and the microtubule-bound RS1, RS2 and the nexin-dynein regulatory complex (N-DRC). From these structures, we identify and build atomic models for 30 proteins, including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor inner dynein arm subform c (IDAc), providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform.

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs.

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

Bis[µ-2-(2-benzoyl-hydrazinylidenemeth-yl)-6-methoxy-phenolato][2-(2-benzoyl-hydrazinylidenemeth-yl)-6-methoxy-phenolato]dimanganese(II) perchlorate methanol solvate.

In the title complex, [Mn(2)(C(15)H(13)N(2)O(3))(3)]ClO(4)·CH(3)OH, the two Mn(II) ions are bridged by two phenolate O atoms from two ligands, forming an Mn(2)O(2) quadrangle. Each Mn(II) ion has a distorted octa-hedral coordination geometry. One Mn(II) ion is coordinated by two N atoms and four O atoms from two ligands, and the other is coordinated by one N atom and five O atoms from three ligands. A dimer is formed by inter-molecular N-H⋯O hydrogen bonds. The dimers, perchlorate anions and methanol solvent mol-ecules are further connected into a chain along [01] through N-H⋯O and O-H⋯O hydrogen bonds.