Structure of human GABAB receptor in an inactive state.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Author Correction: Structure of human GABAB receptor in an inactive state.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure determination by cryoEM at 100 keV.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

Symmetric activation and modulation of the human calcium-sensing receptor.

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

Activation of the TNF-α-Necroptosis Pathway in Parvalbumin-Expressing Interneurons of the Anterior Cingulate Cortex Contributes to Neuropathic Pain.

The hyperexcitability of the anterior cingulate cortex (ACC) has been implicated in the development of chronic pain. As one of the key causes of ACC hyperexcitation, disinhibition of the ACC may be closely related to the dysfunction of inhibitory parvalbumin (PV)-expressing interneurons (PV-INs). However, the molecular mechanism underlying the ACC PV-INs injury remains unclear. The present study demonstrates that spared sciatic nerve injury (SNI) induces an imbalance in the excitation and inhibition (E/I) of the ACC. To test whether tumor necrosis factor-α (TNF-α) upregulation in the ACC after SNI activates necroptosis and participates in PV-INs damage, we performed a differential analysis of transcriptome sequencing using data from neuropathic pain models and found that the expression of genes key to the TNF-α-necroptosis pathway were upregulated. TNF-α immunoreactivity (IR) signals in the ACCs of SNI rats were co-located with p-RIP3- and PV-IR, or p-MLKL- and PV-IR signals. We then systematically detected the expression and cell localization of necroptosis-related proteins, including kinase RIP1, RIP3, MLKL, and their phosphorylated states, in the ACC of SNI rats. Except for RIP1 and MLKL, the levels of these proteins were significantly elevated in the contralateral ACC and mainly expressed in PV-INs. Blocking the ACC TNF-α-necroptosis pathway by microinjecting TNF-α neutralizing antibody or using an siRNA knockdown to block expression of MLKL in the ACC alleviated SNI-induced pain hypersensitivity and inhibited the upregulation of TNF-α and p-MLKL. Targeting TNF-α-triggered necroptosis within ACC PV-INs may help to correct PV-INs injury and E/I imbalance in the ACC in neuropathic pain.

Effects of hCG on DA neuronal death of Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, with the incidence in men being about twice as compared to women. Gender differences may provide clues for finding key targets that mediate the death of dopaminergic (DA) neurons in PD. Luteinizing hormone (LH), analog of human chorionic gonadotropin (hCG), and their receptor, luteinizing hormone/choriogonadotropin receptor (LHCGR), are associated with the pathogenesis of PD. Movement-related symptoms are partially improved by hCG in PD patients. However, the relationship between hCG and PD, as well as its roles in mediating DA neuronal death, has not been elucidated. In this study, we investigated the potential of hCG as a treatment during PD progression. After establishment of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse models, we found that hCG restored the decrease of LHCGR activity caused by down-regulation of LH in the substantia nigra. Furthermore, the reduction of LHCGR activity led to DA neuronal death through knocking down the LHCGR in DA neurons by AAV-mTH-shRNA. Treatment with hCG alleviated the DA neuronal death induced by MPTP. Finally, hCG exerted neuroprotective effects by inhibiting the activation of glycogen synthase kinase 3 beta (GSK3ß) in our MPTP-induced PD mouse and MPP+-treated SH-SY5Y cell models. Together, these results demonstrate that hCG exerts neuroprotective effects for PD through LHCGR, and the inhibition of GSK3ß activation is involved in this protective effect, suggesting that hCG can be taken as a potential therapeutic for the treatment of PD.

Neuroinflammation in the anterior cingulate cortex: the potential supraspinal mechanism underlying the mirror-image pain following motor fiber injury.

BACKGROUND: Peripheral nerve inflammation or lesion can affect contralateral healthy structures, and thus result in mirror-image pain. Supraspinal structures play important roles in the occurrence of mirror pain. The anterior cingulate cortex (ACC) is a first-order cortical region that responds to painful stimuli. In the present study, we systematically investigate and compare the neuroimmune changes in the bilateral ACC region using unilateral- (spared nerve injury, SNI) and mirror-(L5 ventral root transection, L5-VRT) pain models, aiming to explore the potential supraspinal neuroimmune mechanism underlying the mirror-image pain. METHODS: The up-and-down method with von Frey hairs was used to measure the mechanical allodynia. Viral injections for the designer receptors exclusively activated by designer drugs (DREADD) were used to modulate ACC glutamatergic neurons. Immunohistochemistry, immunofluorescence, western blotting, protein microarray were used to detect the regulation of inflammatory signaling. RESULTS: Increased expressions of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and chemokine CX3CL1 in ACC induced by unilateral nerve injury were observed on the contralateral side in the SNI group but on the bilateral side in the L5-VRT group, representing a stronger immune response to L5-VRT surgery. In remote ACC, both SNI and L5-VRT induced robust bilateral increase in the protein level of Nav1.6 (SCN8A), a major voltage-gated sodium channel (VGSC) that regulates neuronal activity in the mammalian nervous system. However, the L5-VRT-induced Nav1.6 response occurred at PO 3d, earlier than the SNI-induced one, 7 days after surgery. Modulating ACC glutamatergic neurons via DREADD-Gq or DREADD-Gi greatly changed the ACC CX3CL1 levels and the mechanical paw withdrawal threshold. Neutralization of endogenous ACC CX3CL1 by contralateral anti-CX3CL1 antibody attenuated the induction and the maintenance of mechanical allodynia and eliminated the upregulation of CX3CL1, TNF-α and Nav1.6 protein levels in ACC induced by SNI. Furthermore, contralateral ACC anti-CX3CL1 also inhibited the expression of ipsilateral spinal c-Fos, Iba1, CD11b, TNF-α and IL-6. CONCLUSIONS: The descending facilitation function mediated by CX3CL1 and its downstream cascade may play a pivotal role, leading to enhanced pain sensitization and even mirror-image pain. Strategies that target chemokine-mediated ACC hyperexcitability may lead to novel therapies for the treatment of neuropathic pain.

Neuroimmune Mechanisms Underlying Neuropathic Pain: The Potential Role of TNF-α-Necroptosis Pathway.

The neuroimmune mechanism underlying neuropathic pain has been extensively studied. Tumor necrosis factor-alpha (TNF-α), a key pro-inflammatory cytokine that drives cytokine storm and stimulates a cascade of other cytokines in pain-related pathways, induces and modulates neuropathic pain by facilitating peripheral (primary afferents) and central (spinal cord) sensitization. Functionally, TNF-α controls the balance between cell survival and death by inducing an inflammatory response and two programmed cell death mechanisms (apoptosis and necroptosis). Necroptosis, a novel form of programmed cell death, is receiving increasing attraction and may trigger neuroinflammation to promote neuropathic pain. Chronic pain is often accompanied by adverse pain-associated emotional reactions and cognitive disorders. Overproduction of TNF-α in supraspinal structures such as the anterior cingulate cortex (ACC) and hippocampus plays an important role in pain-associated emotional disorders and memory deficits and also participates in the modulation of pain transduction. At present, studies reporting on the role of the TNF-α-necroptosis pathway in pain-related disorders are lacking. This review indicates the important research prospects of this pathway in pain modulation based on its role in anxiety, depression and memory deficits associated with other neurodegenerative diseases. In addition, we have summarized studies related to the underlying mechanisms of neuropathic pain mediated by TNF-α and discussed the role of the TNF-α-necroptosis pathway in detail, which may represent an avenue for future therapeutic intervention.

NLRP3-Mediated Piezo1 Upregulation in ACC Inhibitory Parvalbumin-Expressing Interneurons Is Involved in Pain Processing after Peripheral Nerve Injury.

The anterior cingulate cortex (ACC) is particularly critical for pain information processing. Peripheral nerve injury triggers neuronal hyper-excitability in the ACC and mediates descending facilitation to the spinal dorsal horn. The mechanically gated ion channel Piezo1 is involved in the transmission of pain information in the peripheral nervous system. However, the pain-processing role of Piezo1 in the brain is unknown. In this work, we found that spared (sciatic) nerve injury (SNI) increased Piezo1 protein levels in inhibitory parvalbumin (PV)-expressing interneurons (PV-INs) but not in glutaminergic CaMKⅡ+ neurons, in the bilateral ACC. A reduction in the number of PV-INs but not in the number of CaMKⅡ+ neurons and a significant reduction in inhibitory synaptic terminals was observed in the SNI chronic pain model. Further, observation of morphological changes in the microglia in the ACC showed their activated amoeba-like transformation, with a reduction in process length and an increase in cell body area. Combined with the encapsulation of Piezo1-positive neurons by Iba1+ microglia, the loss of PV-INs after SNI might result from phagocytosis by the microglia. In cellular experiments, administration of recombinant rat TNF-α (rrTNF) to the BV2 cell culture or ACC neuron primary culture elevated the protein levels of Piezo1 and NOD-like receptor (NLR) family pyrin domain containing 3 (NLRP3). The administration of the NLRP3 inhibitor MCC950 in these cells blocked the rrTNF-induced expression of caspase-1 and interleukin-1ß (key downstream factors of the activated NLRP3 inflammasome) in vitro and reversed the SNI-induced Piezo1 overexpression in the ACC and alleviated SNI-induced allodynia in vivo. These results suggest that NLRP3 may be the key factor in causing Piezo1 upregulation in SNI, promoting an imbalance between ACC excitation and inhibition by inducing the microglial phagocytosis of PV-INs and, thereby, facilitating spinal pain transmission.

USP49 deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing.

Post-translational histone modifications play important roles in regulating chromatin structure and function. Histone H2B ubiquitination and deubiquitination have been implicated in transcriptional regulation, but the function of H2B deubiquitination is not well defined, particularly in higher eukaryotes. Here we report the purification of ubiquitin-specific peptidase 49 (USP49) as a histone H2B-specific deubiquitinase and demonstrate that H2B deubiquitination by USP49 is required for efficient cotranscriptional splicing of a large set of exons. USP49 forms a complex with RuvB-like1 (RVB1) and SUG1 and specifically deubiquitinates histone H2B in vitro and in vivo. USP49 knockdown results in small changes in gene expression but affects the abundance of >9000 isoforms. Exons down-regulated in USP49 knockdown cells show both elevated levels of alternative splicing and a general decrease in splicing efficiency. Importantly, USP49 is relatively enriched at this set of exons. USP49 knockdown increased H2B ubiquitination (uH2B) levels at these exons as well as upstream 3' and downstream 5' intronic splicing elements. Change in H2B ubiquitination level, as modulated by USP49, regulates U1A and U2B association with chromatin and binding to nascent pre-mRNA. Although H3 levels are relatively stable after USP49 depletion, H2B levels at these exons are dramatically increased, suggesting that uH2B may enhance nucleosome stability. Therefore, this study identifies USP49 as a histone H2B-specific deubiquitinase and uncovers a critical role for H2B deubiquitination in cotranscriptional pre-mRNA processing events.

Poaceae-specific MS1 encodes a phospholipid-binding protein for male fertility in bread wheat.

Male sterility is an essential trait in hybrid seed production for monoclinous crops, including rice and wheat. However, compared with the high percentage of hybrid rice planted in the world, little commercial hybrid wheat is planted globally as a result of the lack of a suitable system for male sterility. Therefore, understanding the molecular nature of male fertility in wheat is critical for commercially viable hybrid wheat. Here, we report the cloning and characterization of Male Sterility 1 (Ms1) in bread wheat by using a combination of advanced genomic approaches. MS1 is a newly evolved gene in the Poaceae that is specifically expressed in microsporocytes, and is essential for microgametogenesis. Orthologs of Ms1 are expressed in diploid and allotetraploid ancestral species. Orthologs of Ms1 are epigenetically silenced in the A and D subgenomes of allohexaploid wheat; only Ms1 from the B subgenome is expressed. The encoded protein, Ms1, is localized to plastid and mitochondrial membranes, where it exhibits phospholipid-binding activity. These findings provide a foundation for the development of commercially viable hybrid wheat.

Upregulation of tumor necrosis factor-alpha in the anterior cingulate cortex contributes to neuropathic pain and pain-associated aversion.

Injury associated pain involves subjective perception and emotional experience. The anterior cingulate cortex (ACC) is a key area involved in the affective component of pain processing. However, the neuroimmune mechanisms underlying enhanced ACC excitability following peripheral nerve injury are still not fully understood. Our previous work has shown that tumor necrosis factor-alpha (TNF-α) overexpression leads to peripheral afferent hyperexcitability and synaptic transmission potentiation in spinal cord. Here, we aimed to reveal the potential role of ACC TNF-α in ACC hyperexcitability and neuropathic pain. c-Fos, a widely used neuronal activity marker, was induced especially in contralateral ACC early [postoperative (PO) 1 h] and later (PO day 7 and 10) during the development of neuropathic pain. Spared nerve injury (SNI) elevated TNF-α level in contralateral ACC from PO day 5 to 14, delayed relative to decreased ipsilateral paw withdrawal threshold apparent from PO day 1 to 14. Microinjection of anti-TNF-α antibody into the ACC completely eliminated c-Fos overexpression and greatly attenuated pain aversion and mechanical allodynia induced by SNI, suggesting an important role of ACC TNF-α in the pain aversiveness and pain maintenance. Furthermore, modulating ACC pyramidal neurons via a Gi-coupled human M4 muscarinic receptor (hM4Di) or a Gq-coupled human M3 muscarinic receptor (hM3Dq), a type of designer receptors exclusively activated by designer drugs (DREADD), greatly changed the ACC TNF-α level and the mechanical paw withdrawal threshold. The positive interactions between TNF-α and ACC neurons might modulate the cytokine microenvironment thus contribute to the neuropathic pain.

Early CALP2 expression and microglial activation are potential inducers of spinal IL-6 up-regulation and bilateral pain following motor nerve injury.

Previous work from our laboratory showed that motor nerve injury by lumbar 5 ventral root transection (L5-VRT) led to interleukin-6 (IL-6) over-expression in bilateral spinal cord, and that intrathecal administration of IL-6 neutralizing antibody delayed the induction of mechanical allodynia in bilateral hind paws. However, early events and upstream mechanisms underlying spinal IL-6 expression following L5-VRT require elucidation. The model of L5-VRT was used to induce neuropathic pain, which was assessed with von Frey hairs and the plantar tester in adult male Sprague-Dawley rats. Calpain-2 (CALP2, a calcium-dependent protease) knockdown or over-expression and microglia depletion were conducted intrathecally. Western blots and immunohistochemistry were performed to explore the possible mechanisms. Here, we provide the first evidence that both IL-6 and CALP2 levels are increased in lumbar spinal cord within 30 min following L5-VRT. IL-6 and CALP2 co-localized in both spinal dorsal horn (SDH) and spinal ventral horn. Post-operative (PO) increase in CALP2 in ipsilateral SDH was evident at 10 min PO, preceding increased IL-6 at 20 min PO. Knockdown of spinal CALP2 by intrathecal CALP2-shRNA administration prevented VRT-induced IL-6 overproduction in ipsilateral spinal cord and alleviated bilateral mechanical allodynia. Spinal microglia activation also played a role in early IL-6 up-regulation. Macrophage/microglia markers ED1/Iba1 were increased at 30 min PO, while glial fibrillary acidic protein (astrocyte) and CNPase (oligodendrocyte) markers were not. Increased Iba1 was detected as early as 20 min PO and peaked at 3 days. Morphology changed from a small soma with fine processes in resting cells to an activated ameboid shape. Depletion of microglia using Mac-1-saporin partially prevented IL-6 up-regulation and attenuated VRT-induced bilateral mechanical allodynia. Taken together, our findings provide evidence that increased spinal cord CALP2 and microglia cell activation may have early causative roles in IL-6 over-expression following motor nerve injury. Agents that inhibit CALP2 and/or microglia activation may therefore prove valuable for treating neuropathic pain.

High-resolution structure of the presynaptic RAD51 filament on single-stranded DNA by electron cryo-microscopy.

Homologous DNA recombination (HR) by the RAD51 recombinase enables error-free DNA break repair. To execute HR, RAD51 first forms a presynaptic filament on single-stranded (ss) DNA, which catalyses pairing with homologous double-stranded (ds) DNA. Here, we report a structure for the presynaptic human RAD51 filament at 3.5-5.0Å resolution using electron cryo-microscopy. RAD51 encases ssDNA in a helical filament of 103Å pitch, comprising 6.4 protomers per turn, with a rise of 16.1Å and a twist of 56.2°. Inter-protomer distance correlates with rotation of an α-helical region in the core catalytic domain that is juxtaposed to ssDNA, suggesting how the RAD51-DNA interaction modulates protomer spacing and filament pitch. We map Fanconi anaemia-like disease-associated RAD51 mutations, clarifying potential phenotypes. We predict binding sites on the presynaptic filament for two modules present in each BRC repeat of the BRCA2 tumour suppressor, a critical HR mediator. Structural modelling suggests that changes in filament pitch mask or expose one binding site with filament-inhibitory potential, rationalizing the paradoxical ability of the BRC repeats to either stabilize or inhibit filament formation at different steps during HR. Collectively, our findings provide fresh insight into the structural mechanism of HR and its dysregulation in human disease.

CTF Challenge: Result summary.

Image formation in bright field electron microscopy can be described with the help of the contrast transfer function (CTF). In this work the authors describe the "CTF Estimation Challenge", called by the Madrid Instruct Image Processing Center (I2PC) in collaboration with the National Center for Macromolecular Imaging (NCMI) at Houston. Correcting for the effects of the CTF requires accurate knowledge of the CTF parameters, but these have often been difficult to determine. In this challenge, researchers have had the opportunity to test their ability in estimating some of the key parameters of the electron microscope CTF on a large micrograph data set produced by well-known laboratories on a wide set of experimental conditions. This work presents the first analysis of the results of the CTF Estimation Challenge, including an assessment of the performance of the different software packages under different conditions, so as to identify those areas of research where further developments would be desirable in order to achieve high-resolution structural information.

Calpain-2 contributes to neuropathic pain following motor nerve injury via up-regulating interleukin-6 in DRG neurons.

Motor nerve injury by L5 ventral root transection (L5-VRT) initiates interleukin-6 (IL-6) up-regulation in primary afferent system contributing to neuropathic pain. However, the early upstream regulatory mechanisms of IL-6 after L5-VRT are still unknown. Here, we monitored both the activity of calpain, a calcium-dependent protease suggested as one of the earliest mediators for cytokine regulation, and the expression of IL-6 in bilateral L4-L6 dorsal root ganglias (DRGs) soon after L5-VRT. We found that the protein level of calpain-2 in DRGs, but not calpain-1 was increased transiently in the first 10 min(-1)h ipsilaterally and 20 min(-1)h contralaterally after L5-VRT, long before mechanical allodynia was initiated (5-15 h ipsilaterally and 15 h(-1)d contralaterally). The early activation of calpain evaluated by the generation of spectrin breakdown products (SBDP) correlated well with IL-6 up-regulation in bilateral DRGs. Double immunofluorescence staining revealed that almost all the calpain-2 positive neurons expressed IL-6, indicating an association between calpain-2 and IL-6. Inhibition of calpain by pre-treatment with MDL28170 (25mg/kg, i.p.) attenuated the rat mechanical allodynia and prevented the early up-regulation of IL-6 following L5-VRT. Addition of exogenous calpain-2 onto the surface of left L5 DRG triggered a temporal allodynia and increased IL-6 in bilateral DRGs simultaneously. Taken together, the early increase of calpain-2 in L5-VRT rats might be responsible for the induction of allodynia via up-regulating IL-6 in DRG neurons.

The BRC repeats of human BRCA2 differentially regulate RAD51 binding on single- versus double-stranded DNA to stimulate strand exchange.

The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA recombination (HDR) to preserve genome stability. BRCA2 binds to RAD51 through 8 conserved BRC repeat motifs dispersed in an 1127-residue region (BRCA2([BRC1-8])). Here, we show that BRCA2([BRC1-8]) exerts opposing effects on the binding of RAD51 to single-stranded (ss) versus double-stranded (ds) DNA substrates, enhancing strand exchange. BRCA2([BRC1-8]) alters the electrophoretic mobility of RAD51 bound to an ssDNA substrate, accompanied by an increase in ssDNA-bound protein assemblies, revealed by electron microscopy. Single-molecule fluorescence spectroscopy shows that BRCA2([BRC1-8]) promotes RAD51 loading onto ssDNA. In contrast, BRCA2([BRC1-8]) has a different effect on RAD51 assembly on dsDNA; it suppresses and slows this process. When homologous ssDNA and dsDNA are both present, BRCA2([BRC1-8]) stimulates strand exchange, with delayed RAD51 loading onto dsDNA accompanying the appearance of joint molecules representing recombination products. Collectively, our findings suggest that BRCA2([BRC1-8]) targets RAD51 to ssDNA while inhibiting dsDNA binding and that these contrasting activities together bolster one another to stimulate HDR. Our work provides fresh insight into the mechanism of HDR in humans, and its regulation by the BRCA2 tumor suppressor.

Interaction of human erythrocyte catalase with air-water interface in cryoEM.

One of the key goals in single-particle cryo-microscopy is to obtain a uniform distribution of particle orientations, so that the three-dimensional structure has isotropic resolution in Fourier space. A common problem arises from the interaction of protein molecules with the air-water interface that exists on both surfaces of the thin film of liquid that is formed prior to plunge-freezing into liquid ethane. Some proteins and other macromolecular complexes are disrupted by interaction with the air-water interface. Other proteins or macromolecules either become concentrated through their interaction with the interface or are excluded because they bind strongly to some other part of the grid or the filter paper used in blotting. In this paper, the interaction of human erythrocyte catalase with the air-water interface is investigated and minimized by the addition of certain detergents. Detergents can form an amphipathic monolayer at the air-water interface that creates a barrier and leaves the molecules free to adopt a variety of orientations, thus facilitating the 3D structure determination. These results suggest that further characterization and development of detergents for cryo-microscopy plunge-freezing would be useful.

Association of Peripheral Blood Biomarkers With Response to Anti-PD-1 Immunotherapy for Patients With Deficient Mismatch Repair Metastatic Colorectal Cancer: A Multicenter Cohort Study.

Purpose: Deficient mismatch repair (dMMR) is an established biomarker for the response to the programmed cell death (PD)-1 inhibitors in metastatic colorectal cancer (mCRC). Although patients with dMMR mCRC could achieve a high incidence of disease control and favorable progression-free survival (PFS), reported response rates to PD-1 inhibitors are variable from 28% to 52%. We aimed to explore the additional predictive biomarkers associated with response to anti-PD-1 immunotherapy in patients with dMMR mCRC. Methods: This multicenter cohort study enrolled patients with dMMR mCRC receiving anti-PD-1 immunotherapy at the Sixth Affiliated Hospital of Sun Yat-sen University and Sun Yat-sen University Cancer Center between December 2016 and December 2019. The total information of 20 peripheral blood biomarkers, including T cells (frequency of CD4+ T cell, frequency of CD8+ T cell, and ratio of CD4+/CD8+), carcinoembryonic antigen (CEA), inflammatory markers, and lipid metabolism markers, was collected. The association between response or survival and peripheral blood parameters was analyzed. Results: Among the tested parameters, the ratio of CD4+/CD8+ and frequency of CD4+ T cell were significantly associated with PFS (p = 0.023, p = 0.012) and overall survival (OS; p = 0.027, p = 0.019) in a univariate analysis. A lower level of CD4+/CD8+ ratio or frequency of CD4+ T cell showed a significant association with better overall response rates (ORRs; p = 0.03, p = 0.01). The ratio of CD4+/CD8+ and frequency of CD4+ T cell maintained significance in multivariate Cox model for PFS (HR = 9.23, p = 0.004; HR = 4.83, p = 0.02) and OS (HR = 15.22, p = 0.009; HR = 16.21, p = 0.025). Conclusion: This study indicated that the ratio of CD4+/CD8+ and the frequency of CD4+ T cell might be crucial independent biomarkers within dMMR mCRC to better identify patients for anti-PD-1 immunotherapy. If validated in prospective clinical trials, the ratio of CD4+/CD8+ and the frequency of CD4+ T cell might aid in guiding the treatment of PD-1 inhibitors among patients with dMMR mCRC.

Three-dimensional reconstruction of Heterocapsa circularisquama RNA virus by electron cryo-microscopy.

Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major natural agent controlling the host population. The viral capsid is constructed from a single gene product. Electron cryo-microscopy revealed that the virus has a diameter of 34 nm and T = 3 symmetry. The 180 quasi-equivalent monomers have an unusual arrangement in that each monomer contributes to a 'bump' on the surface of the protein. Though the capsid protein probably has the classic 'jelly roll' ß-sandwich fold, this is a new packing arrangement and is distantly related to the other positive-sense ssRNA virus capsid proteins. The handedness of the structure has been determined by a novel method involving high resolution scanning electron microscopy of the negatively stained viruses and secondary electron detection.