TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile.

The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.

An Atomic Structure of the Human Spliceosome.

Mechanistic understanding of pre-mRNA splicing requires detailed structural information on various states of the spliceosome. Here we report the cryo electron microscopy (cryo-EM) structure of the human spliceosome just before exon ligation (the C∗ complex) at an average resolution of 3.76 Å. The splicing factor Prp17 stabilizes the active site conformation. The step II factor Slu7 adopts an extended conformation, binds Prp8 and Cwc22, and is poised for selection of the 3'-splice site. Remarkably, the intron lariat traverses through a positively charged central channel of RBM22; this unusual organization suggests mechanisms of intron recruitment, confinement, and release. The protein PRKRIP1 forms a 100-Å α helix linking the distant U2 snRNP to the catalytic center. A 35-residue fragment of the ATPase/helicase Prp22 latches onto Prp8, and the quaternary exon junction complex (EJC) recognizes upstream 5'-exon sequences and associates with Cwc22 and the GTPase Snu114. These structural features reveal important mechanistic insights into exon ligation.

Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

Structural basis of pre-tRNA intron removal by human tRNA splicing endonuclease.

Removal of the intron from precursor-tRNA (pre-tRNA) is essential in all three kingdoms of life. In humans, this process is mediated by the tRNA splicing endonuclease (TSEN) comprising four subunits: TSEN2, TSEN15, TSEN34, and TSEN54. Here, we report the cryo-EM structures of human TSEN bound to full-length pre-tRNA in the pre-catalytic and post-catalytic states at average resolutions of 2.94 and 2.88 Å, respectively. Human TSEN features an extended surface groove that holds the L-shaped pre-tRNA. The mature domain of pre-tRNA is recognized by conserved structural elements of TSEN34, TSEN54, and TSEN2. Such recognition orients the anticodon stem of pre-tRNA and places the 3'-splice site and 5'-splice site into the catalytic centers of TSEN34 and TSEN2, respectively. The bulk of the intron sequences makes no direct interaction with TSEN, explaining why pre-tRNAs of varying introns can be accommodated and cleaved. Our structures reveal the molecular ruler mechanism of pre-tRNA cleavage by TSEN.

Mechanism of exon ligation by human spliceosome.

Pre-mRNA splicing involves two sequential reactions: branching and exon ligation. The C complex after branching undergoes remodeling to become the C∗ complex, which executes exon ligation. Here, we report cryo-EM structures of two intermediate human spliceosomal complexes, pre-C∗-I and pre-C∗-II, both at 3.6 Å. In both structures, the 3' splice site is already docked into the active site, the ensuing 3' exon sequences are anchored on PRP8, and the step II factor FAM192A contacts the duplex between U2 snRNA and the branch site. In the transition of pre-C∗-I to pre-C∗-II, the step II factors Cactin, FAM32A, PRKRIP1, and SLU7 are recruited. Notably, the RNA helicase PRP22 is positioned quite differently in the pre-C∗-I, pre-C∗-II, and C∗ complexes, suggesting a role in 3' exon binding and proofreading. Together with information on human C and C∗ complexes, our studies recapitulate a molecular choreography of the C-to-C∗ transition, revealing mechanistic insights into exon ligation.

A phytoplasma effector destabilizes chloroplastic glutamine synthetase inducing chlorotic leaves that attract leafhopper vectors.

Leaf yellowing is a well-known phenotype that attracts phloem-feeding insects. However, it remains unclear how insect-vectored plant pathogens induce host leaf yellowing to facilitate their own transmission by insect vectors. Here, we report that an effector protein secreted by rice orange leaf phytoplasma (ROLP) inhibits chlorophyll biosynthesis and induces leaf yellowing to attract leafhopper vectors, thereby presumably promoting pathogen transmission. This effector, designated secreted ROLP protein 1 (SRP1), first secreted into rice phloem by ROLP, was subsequently translocated to chloroplasts by interacting with the chloroplastic glutamine synthetase (GS2). The direct interaction between SRP1 and GS2 disrupts the decamer formation of the GS2 holoenzyme, attenuating its enzymatic activity, thereby suppressing the synthesis of chlorophyll precursors glutamate and glutamine. Transgenic expression of SRP1 in rice plants decreased GS2 activity and chlorophyll precursor accumulation, finally inducing leaf yellowing. This process is correlated with the previous evidence that the knockout of GS2 expression in rice plants causes a similar yellow chlorosis phenotype. Consistently, these yellowing leaves attracted higher numbers of leafhopper vectors, caused the vectors to probe more frequently, and presumably facilitate more efficient phytoplasma transmission. Together, these results uncover the mechanism used by phytoplasmas to manipulate the leaf color of infected plants for the purpose of enhancing attractiveness to insect vectors.

THUMPD2 catalyzes the N2-methylation of U6 snRNA of the spliceosome catalytic center and regulates pre-mRNA splicing and retinal degeneration.

The mechanisms by which the relatively conserved spliceosome manages the enormously large number of splicing events that occur in humans (∼200 000 versus ∼300 in yeast) are poorly understood. Here, we show deposition of one RNA modification-N2-methylguanosine (m2G) on the G72 of U6 snRNA (the catalytic center of the spliceosome) promotes efficient pre-mRNA splicing activity in human cells. This modification was identified to be conserved among vertebrates. Further, THUMPD2 was demonstrated as the methyltransferase responsible for U6 m2G72 by explicitly recognizing the U6-specific sequences and structural elements. The knock-out of THUMPD2 eliminated U6 m2G72 and impaired the pre-mRNA splicing activity, resulting in thousands of changed alternative splicing events of endogenous pre-mRNAs in human cells. Notably, the aberrantly spliced pre-mRNA population elicited the nonsense-mediated mRNA decay pathway. We further show that THUMPD2 was associated with age-related macular degeneration and retinal function. Our study thus demonstrates how an RNA epigenetic modification of the major spliceosome regulates global pre-mRNA splicing and impacts physiology and disease.

The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1R1074H and PHF5AY36C The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5AR38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

Complanatuside A ameliorates 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice by regulating the Th17/Treg balance via the JAK2/STAT3 signaling pathway.

Immunity imbalance of T helper 17 (Th17)/regulatory T (Treg) cells is involved in the pathogenesis of Crohn's disease (CD). Complanatuside A (CA), a flavonol glycoside, exerts anti-inflammatory activities and our study aimed to identify its effect on TNBS-induced colitis and the possible mechanisms. We found that CA alleviated the symptoms of colitis in TNBS mice, as demonstrated by prevented weight loss and colon length shortening, as well as decreased disease activity index scores, inflammatory scores, and levels of proinflammatory factors. Flow cytometry analysis showed that CA markedly reduced the percentage of Th17 cells while increasing the percentage of Treg cells in TNBS mice. Under Th17 cell polarizing conditions, CA inhibited the differentiation of Th17 cells while the Treg cell differentiation was elevated under Treg cell polarizing conditions. Furthermore, it was observed that JAK2 interacted with CA through six hydrogen bonds via molecular docking. The phosphorylation of JAK2/STAT3 was reduced by CA, which might be correlated with the protective effect of CA on colitis. In conclusion, CA reduced the imbalance of Th17/Treg cells by inhibiting the JAK2/STAT3 signaling pathway in TNBS-induced colitis, which may provide novel strategies for CD treatment.

Brain extended and closed forms glutathione levels decrease with age and extended glutathione is associated with visuospatial memory.

During aging, the brain is subject to greater oxidative stress (OS), which is thought to play a critical role in cognitive impairment. Glutathione (GSH), as a major antioxidant in the brain, can be used to combat OS. However, how brain GSH levels vary with age and their associations with cognitive function is unclear. In this study, we combined point-resolved spectroscopy and edited spectroscopy sequences to investigate extended and closed forms GSH levels in the anterior cingulate cortex (ACC), posterior cingulate cortex (PCC), and occipital cortex (OC) of 276 healthy participants (extended form, 166 females, age range 20-70 years) and 15 healthy participants (closed form, 7 females, age range 26-56 years), and examined their relationships with age and cognitive function. The results revealed decreased extended form GSH levels with age in the PCC among 276 participants. Notably, the timecourse of extended form GSH level changes in the PCC and ACC differed between males and females. Additionally, positive correlations were observed between extended form GSH levels in the PCC and OC and visuospatial memory. Additionally, a decreased trend of closed form GSH levels with age was also observed in the PCC among 15 participants. Taken together, these findings enhance our understanding of the brain both closed and extended form GSH time course during normal aging and associations with sex and memory, which is an essential first step for understanding the neurochemical underpinnings of healthy aging.

Silver-Enabled Dearomative Trifluoromethoxylation of Indoles.

The only known method for the dearomative trifluoromethoxylation of indoles is preliminary, with only one substrate successfully undergoing the reaction. In this study, we not only developed a broadly applicable method for indole dearomative trifluoromethoxylation but also achieved divergent trifluoromethoxylation by fine-tuning the reaction conditions. Under optimized conditions, with a silver salt and an easily handled OCF3 reagent, various indoles smoothly underwent dearomatization to afford a diverse array of ditrifluoromethoxylated indolines in 50-84% isolated yields with up to 37:1 diastereoselectivity, and fluorinated trifluoromethoxylated indolines were obtained with exclusive trans selectivity. In addition, the reaction conditions were compatible with other heteroaromatic rings as well as styrene moieties.

A nonstructural protein encoded by a rice reovirus induces an incomplete autophagy to promote viral spread in insect vectors.

Viruses can hijack autophagosomes as the nonlytic release vehicles in cultured host cells. However, how autophagosome-mediated viral spread occurs in infected host tissues or organs in vivo remains poorly understood. Here, we report that an important rice reovirus, rice gall dwarf virus (RGDV) hijacks autophagosomes to traverse multiple insect membrane barriers in the midgut and salivary gland of leafhopper vector to enhance viral spread. Such virus-containing double-membraned autophagosomes are prevented from degradation, resulting in increased viral propagation. Mechanistically, viral nonstructural protein Pns11 induces autophagy and embeds itself in the autophagosome membranes. The autophagy-related protein 5 (ATG5)-ATG12 conjugation is essential for initial autophagosome membrane biogenesis. RGDV Pns11 specifically interacts with ATG5, both in vitro and in vivo. Silencing of ATG5 or Pns11 expression suppresses ATG8 lipidation, autophagosome formation, and efficient viral propagation. Thus, Pns11 could directly recruit ATG5-ATG12 conjugation to induce the formation of autophagosomes, facilitating viral spread within the insect bodies. Furthermore, Pns11 potentially blocks autophagosome degradation by directly targeting and mediating the reduced expression of N-glycosylated Lamp1 on lysosomal membranes. Taken together, these results highlight how RGDV remodels autophagosomes to benefit viral propagation in its insect vector.

Huogu injection protects against SONFH by promoting osteogenic differentiation of BMSCs and preventing osteoblast apoptosis.

To investigate the effect and mechanism of Huogu injection (HG) on steroid-induced osteonecrosis of the femoral head (SONFH), we established a SONFH model in rabbits using horse serum and dexamethasone (DEX) and applied HG locally at the hip joint. We evaluated the therapeutic efficacy at 4 weeks using scanning electron microscopy (SEM), micro-CT, and qualitative histology including H&E, Masson's trichrome, ALP, and TUNEL staining. In vitro, we induced osteogenic differentiation of bone marrow stromal cells (BMSCs) and performed analysis on days 14 and 21 of cell differentiation. The findings, in vivo, including SEM, micro-CT, and H&E staining, showed that HG significantly maintained bone quality and trabecular number. ALP staining indicated that HG promoted the proliferation of bone cells. Moreover, the results of Masson's trichrome staining demonstrated the essential role of HG in collagen synthesis. Additionally, TUNEL staining revealed that HG reduced apoptosis. ALP and ARS staining in vitro confirmed that HG enhanced osteogenic differentiation and mineralization, consistent with the WB and qRT-PCR analysis. Furthermore, Annexin V-FITC/PI staining verified that HG inhibited osteoblast apoptosis, in agreement with the WB and qRT-PCR analyses. Furthermore, combined with the UPLC analysis, we found that naringin enhanced the osteogenic differentiation and accelerated the deposition of calcium phosphate. Salvianolic acid B protected osteoblasts derived from BMSCs against GCs-mediated apoptosis. Thus, this study not only reveals the mechanism of HG in promoting osteogenesis and anti-apoptosis of osteoblasts but also identifies the active-related components in HG, by which we provide the evidence for the application of HG in SONFH.

Tetratricopeptide-containing SMALL KERNEL 11 is essential for the assembly of cytochrome c oxidase in maize mitochondria.

Assembly of the functional complexes of the mitochondrial respiratory chain requires sophisticated and efficient regulatory mechanisms. In plants, the subunit composition and assembly factors involved in the biogenesis of cytochrome c oxidase (complex IV) are substantially less defined than in mammals and yeast. In this study, we cloned maize (Zea mays) Small kernel 11 (Smk11) via map-based cloning. Smk11 encodes a mitochondria-localized tetratricopeptide repeat protein. Disruption of Smk11 severely affected the assembly and activity of mitochondrial complex IV, leading to delayed plant growth and seed development. Protein interactions studies revealed that SMK11 might interact with four putative complex IV assembly factors, Inner membrane peptidase 1A (ZmIMP1A), MYB domain protein 3R3 (ZmMYB3R-3), cytochrome c oxidase 23 (ZmCOX23), and mitochondrial ferredoxin 1 (ZmMFDX1), among which ZmMFDX1 might interact with subunits ZmCOX6a and ZmCOX-X1; ZmMYB3R-3 might also interact with ZmCOX6a. The mutation of SMK11 perturbed the normal assembly of these subunits, leading to the inactivation of complex IV. The results of this study revealed that SMK11 serves as an accessory assembly factor required for the normal assembly of subunits into complex IV, which will accelerate the elucidation of the assembly of complex IV in plant mitochondria.

Diversity-Oriented Synthesis of Indole-Fused Polycyclic Scaffolds via Rhodium-Catalyzed NH-Indole-Directed C-H Coupling of 2-Phenyl-1H-indoles with Propargylic Alcohol Derivatives.

Diversity-oriented synthesis strategy for the efficient assembly of indole-fused polycyclic scaffolds via rhodium-catalyzed NH-indole-directed C-H coupling with propargylic alcohol derivatives in a regioselective manner was developed. Five 2-phenyl-1H-indole-embedded core skeletons were synthesized. In particular, three different indole-fused exo-olefin-containing polycycles were realized, which may be manipulated for further chemistry.

Cascade Electrochemical Aerobic Oxygenation of 2-Substituted Indoles and Electrochemical [5 + 3] Annulation with Amidines: Access to Eight-Membered Benzo[1,3,5]triazocin-6(5H)-ones.

The cascade electrochemical C3-selective aerobic oxygenation of 2-substituted indoles and electrochemical [5 + 3] annulation with amidines through an undivided cell galvanostatic method employing molecular oxygen and "electricity" as green oxidants was developed. This protocol provides an efficient and direct approach to eight-membered benzo[1,3,5]triazocin-6(5H)-ones. Mechanistic studies suggested that two subsequent electrochemical processes both proceeded through radical pathways.

Association of sleep traits with myopia in children and adolescents: A meta-analysis and Mendelian randomization study.

PURPOSE: The association between sleep and myopia in children and adolescents has been reported, yet it remains controversial and inconclusive. This study aimed to investigate the influence of different sleep traits on the risk of myopia using meta-analytical and Mendelian randomization (MR) techniques. METHODS: The literature search was performed in August 31, 2023 based on PubMed, Embase, Web of Science, and Cochrane library. The meta-analysis of observational studies reporting the relationship between sleep and myopia was conducted. MR analyses were carried out to assess the causal impact of genetic pre-disposition for sleep traits on myopia. RESULTS: The results of the meta-analysis indicated a significant association between the risk of myopia and both short sleep duration [odds ratio (OR) = 1.23, 95% confidence interval (CI) = 1.08-1.42, P = 0.003] and long sleep duration (OR = 0.75, 95% CI = 0.66-0.86, P < 0.001). MR analyses revealed no significant causal associations of genetically determined sleep traits with myopia, including chronotype, sleep duration, short sleep duration and long sleep duration (all P > 0.05). CONCLUSIONS: No evidence was found to support a causal relationship between sleep traits and myopia. While sleep may not independently predict the risk of myopia, the potential impact of sleep on the occurrence and development of myopia cannot be disregarded.

Effect of Premedication With Pronase Before Upper Gastrointestinal Endoscopy: A Multicenter Prospective Randomized Controlled Study.

OBJECTIVES: This study aimed to confirm whether premedication with pronase before endoscopy improves mucosal visualization and increases precancerous lesion and cancer lesion detection rates. MATERIALS AND METHODS: From June 2018 to April 2019, out-patients scheduled for endoscopy from 13 hospitals were screened to be randomly allocated in a 2:1 ratio to premedication with pronase (group A) and water (group B). The primary endpoint was mucosal visibility scores, and the secondary endpoint was precancerous and cancer lesion detection rates. This trial was registered at Chinese Clinical Trial Registry, and the registration number was ChiCTR1800016853. RESULTS: Group A showed significantly lower mucosal visibility scores (better mucosal visibility) of esophagus, stomach, and duodenum than group B, with all P -values <0.001. The overall cancer detection rates between group A and group B were 0.83 and 1.08%, and overall detection rates of precancerous and cancer lesion were 4.4 and 4.9%, both without significant difference ( P =1.000 and 0.824). In addition, the flushing volume (milliliter) of group A (10.52±23.41) was less than group B (36.30±52.11) ( P <0.001), and the flushing frequency of group A (0.46±1.01) was fewer than group B (1.62±2.12) ( P <0.001). CONCLUSIONS: Premedication with pronase could achieve better mucosal visibility and decrease flushing frequency and volume, but may not increase lesion detection rates.

A multiplex digital PCR assay for detection and quantitation of porcine circovirus type 2 and type 3.

Porcine circovirus (PCV) has become a major pathogen, causing major economic losses in the global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) are distributed worldwide. We designed specific primer and probe sequences targeting PCV2 Cap and PCV3 Rap and developed a multiplex crystal digital PCR (cdPCR) method after optimizing the primer concentration, probe concentration, and annealing temperature. The multiplex cdPCR assay permits precise and differential detection of PCV2 and PCV3, with a limit of detection of 1.39 × 101 and 1.27 × 101 copies/reaction, respectively, and no cross-reaction with other porcine viruses was observed. The intra-assay and interassay coefficients of variation (CVs) were less than 8.75%, indicating good repeatability and reproducibility. To evaluate the practical value of this assay, 40 tissue samples and 70 feed samples were tested for both PCV2 and PCV3 by cdPCR and quantitative PCR (qPCR). Using multiplex cdPCR, the rates of PCV2 infection, PCV3 infection, and coinfection were 28.45%, 1.72%, and 12.93%, respectively, and using multiplex qPCR, they were 25.00%, 0.86%, and 4.31%, respectively This highly specific and sensitive multiplex cdPCR thus allows accurate simultaneous detection of PCV2 and PCV3, and it is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrices.

Endoscopic treatment for early duodenal papillary carcinoma: long-term outcomes.

BACKGROUND AND AIM: This study aims to determine whether endoscopic papillectomy (EP) is a safe and effective treatment for early duodenal papillary carcinoma with long-term follow-up. METHODS: From June 2012 to September 2022, 48 patients with early duodenal papilloma carcinoma who received endoscopic treatment were included. The histological types, percentage of complete resections, postoperative residuals, adverse events, and recurrences were evaluated. RESULTS: EP was successful in all patients; 46 were lumped, and two were fragmented, with a 95.8% intact removal rate (46/48). The preoperative biopsy pathological positive rate was 70.8% (34/48). The incidence of early postoperative adverse events (within 1 month after EP) were 16.7% (8/48), including four cases of acute pancreatitis, three cases of delayed bleeding, and one case of acute cholangitis. In addition, 4.2% (2/48) of the late adverse events were bile duct stenosis. After 6 months, the postoperative residual rate was 0%. The median time to recurrence was 17.5 months, and the postoperative recurrence rate was 16.7% (8/48) in patients treated with radiofrequency ablation. The median progression-free survival was 18.6 months (95% CI, 12.1-25.1), and the median overall survival was 121.5 months (95% CI, 105.6-120.9). CONCLUSIONS: EP is a safe and efficient alternative therapy for early duodenal papillary carcinoma. Endoscopic follow-up and treatment are essential because of the potential for recurrence.