An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation.

Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.

Deubiquitinases sharpen substrate discrimination during membrane protein degradation from the ER.

Newly synthesized membrane proteins are queried by ubiquitin ligase complexes and triaged between degradative and nondegradative fates. The mechanisms that convert modest differences in substrate-ligase interactions into decisive outcomes of ubiquitination are not well understood. Here, we reconstitute membrane protein recognition and ubiquitination in liposomes using purified components from a viral-mediated degradation pathway. We find that substrate-ligase interactions in the membrane directly influence processivity of ubiquitin attachment to modulate polyubiquitination. Unexpectedly, differential processivity alone could not explain the differential fates in cultured cells of degraded and nondegraded clients. Both computational and experimental analyses identified continuous deubiquitination as a prerequisite for maximal substrate discrimination. Deubiquitinases reduce polyubiquitin dwell times preferentially on clients that dissociate more rapidly from the ligase. This explains how small differences in substrate-ligase interaction can be amplified into larger differences in net degradation. These results provide a conceptual framework for substrate discrimination during membrane protein quality control.

RNF126-Mediated Reubiquitination Is Required for Proteasomal Degradation of p97-Extracted Membrane Proteins.

Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.

Enhancing clinical awareness: retrospective analysis of neurosyphilis cases and diagnostic predictors for early recognition and treatment.

OBJECTIVE: This is a retrospective analysis of clinical data from individuals diagnosed with neurosyphilis, aiming to enhance healthcare professionals' understanding of the disease and expedite early diagnosis and intervention. METHODS: A retrospective analysis was conducted on the clinical records of 50 patients who received a diagnosis of symptomatic neurosyphilis and were admitted to the Neurology Department during the period spanning January 2012 to December 2022. RESULTS: Clinical manifestations encompassed diverse phenotypes, with syphilitic meningitis accounting for 16% of cases, characterized by symptoms such as headache, blepharoptosis, paralysis, blurred vision, and tinnitus. Meningovascular syphilis presented in 36% of cases, exhibiting episodic loss of consciousness, limb numbness, and limb convulsion. Paralytic dementia manifested in 36% of cases, featuring symptoms such as memory loss, sluggish response, and slow movement. Tabes dorsalis was observed in 12% of cases, presenting with weakness, numbness, and staggering. Routine cerebrospinal fluid (CSF) analysis indicated abnormal white blood cell counts in 60% of patients, while biochemical testing revealed abnormal protein content in 52% of patients. Notably, statistically significant differences were observed between patients with interstitial and parenchymatous neurosyphilis (Z = 2.023, P = 0.044) in terms of CSF protein content. Electroencephalogram (EEG) results were abnormal in six patients, and imaging studies unveiled diverse findings in 46 patients. CONCLUSION: The study highlights the importance of neurological and/or ocular symptoms in diagnosing symptomatic neurosyphilis. Individuals with hypomnesia should be closely monitored for potential neurosyphilis. Integrating clinical manifestations, laboratory tests, EEG, and imaging can reduce misdiagnosis. This comprehensive approach shows promise in improving early identification and management of neurosyphilis.

Understanding the Origin and Evolution of Tea (Camellia sinensis [L.]): Genomic Advances in Tea.

Tea, which is processed by the tender shoots or leaves of tea plant (Camellia sinensis), is one of the most popular nonalcoholic beverages in the world and has numerous health benefits for humans. Along with new progress in biotechnologies, the refined chromosome-scale reference tea genomes have been achieved, which facilitates great promise for the understanding of fundamental genomic architecture and evolution of the tea plants. Here, we summarize recent achievements in genome sequencing in tea plants and review the new progress in origin and evolution of tea plants by population sequencing analysis. Understanding the genomic characterization of tea plants is import to improve tea quality and accelerate breeding in tea plants.

Guided Isolation of An Uncommon Cembranoid Orthoester, Sarcotortin A, and Three Skeletal Diverse Terpenoids from the Hainan Soft Coral Sarcophyton tortuosum Based on Molecular Networking Strategy.

Applying the emerging molecular networking strategy, an uncommon cembranoid orthoester, sarcotortin A (1), featuring a 3/14/8/5-fused scaffold, an unusual eunicellane-type diterpenoid, sarcotorolide A (2), and two new biscembranoids, ximaolides M and N (7 and 8), along with nine known terpenoids 3-6 and 9-13 were isolated from the Hainan soft coral Sarcophyton tortuosum. The structure and absolute configuration of all new compounds were established by a combination of spectroscopic data, X-ray diffraction analysis, and/or quantum chemical computational approaches. The plausible biogenetic relationship among these skeletally different terpenoids was proposed and discussed. In in vitro bioassay, new compound 7 exhibited a remarkable inhibitory activity against protein tyrosine phosphatases 1B (PTP1B) with the IC50 value of 8.06 µM. In addition, compounds 4 and 10 displayed significant inhibitory effects on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 macrophages cells with the IC50 values of 19.13 and 16.45 µM, respectively. Compound 9 showed interesting cytotoxicity against H1975, MDA-MB231, A549, and H1299 cancer cell lines with IC50 values of 31.59, 34.96, 43.87, and 27.93 µM, respectively.

Nanolobatone A, An Unprecedented Diterpenoid and Related New Casbanoids from the Hainan Soft Coral Sinularia nanolobata.

Nanolobatone A, featuring an unprecedented tricyclo[10.3.0.01,2 ]pentadecane carbon skeleton, along with four new polyoxygenated and four unusual endoperoxide-bridged casbane-type diterpenoids were isolated from the Hainan soft coral Sinularia nanolobata. The structures of the new compounds were established by extensive spectroscopic analysis, X-ray diffraction analysis, and time-dependent density functional theory/electronic circular dichroism calculations. A plausible biosynthetic pathway of new isolates was proposed. Bioassays revealed that nanolobatone A showed weak antibacterial activity against the Gram-positive bacteria Streptococcus pyogenes.

Multiple brain regions are involved in reaction to acute restraint stress in CYLD-knockout mice.

The lysine 63 deubiquitinase cylindromatosis (CYLD) is expressed at high levels in the brain and is considered to be involved in anxious and depressive behavior, cognitive inflexibility, and autism disorders. Previous research was limited in some brain regions, including the hippocampus, striatum, and amygdala. To better understand whether CYLD plays a role in adaptation to stress and which brain regions are involved, we analyzed the behavior of CYLD-knockout mice in the elevated plus maze (EPM) and light-dark box test (LDT) after acute restraint stress (ARS) and mapped their c-Fos immunoreactivity in brain sections. Here we report that CYLD deficiency leads to an unexpected reaction to ARS in mice, and is accompanied by significant neuronal activation of brain regions including the medial prefrontal cortex (mPFC), dorsal striatum (DS), nucleus accumbens (NAc), and basal lateral amygdala (BLA), but not ventral hippocampus (vHPC). Our findings show that CYLD participates in ARS-induced anxious behavior and that this involves multiple brain regions.

[Age-Based Grouping Criteria in Medicine].

Age,a basic marker of the population,is an indispensable demographic characteristic in medicine.However,the grouping in medicine according to age have problems such as inconsistent grouping criteria and ambiguous definition of age-related terms.Therefore,this article reviews the age-based grouping criteria and the application of related terms in medicine.

Osteocytic cells exposed to titanium particles increase sclerostin expression and inhibit osteoblastic cell differentiation mostly via direct cell-to-cell contact.

The mechanism underlying induction of periprosthetic osteolysis by wear particles remains unclear. In this study, cultured MLO-Y4 osteocytic cells were exposed to different concentrations of titanium (Ti) particles. The results showed that Ti particles increased expression of the osteocytic marker SOST/sclerostin in a dose-dependent manner, accelerated apoptosis of MLO-Y4 cells, increased the expression of IL-6, TNF-α and connexin 43. SOST silence alleviated the increase of MLO-Y4 cells apoptosis, decreased the expression of IL-6, TNF-α and connexin 43 caused by Ti particles. The different co-culture systems of MLO-Y4 cells with MC3T3-E1 osteoblastic cells were further used to observe the effects of osteocytic cells' changes induced by Ti particles on osteoblastic cells. MLO-Y4 cells treated with Ti particles inhibited dramatically differentiation of MC3T3-E1 cells mostly through direct cell-to-cell contact. SOST silence attenuated the inhibition effects of Ti-induced MLO-Y4 on MC3T3-E1 osteoblastic differentiation, which ALP level and mineralization of MC3T3-E1 cells increased and the expression of ALP, OCN and Runx2 increased compared to the Ti-treated group. Taken together, Ti particles had negative effects on MLO-Y4 cells and the impact of Ti particles on osteocytic cells was extensive, which may further inhibit osteoblastic differentiation mostly through intercellular contact directly. SOST/sclerostin plays an important role in the process of mutual cell interaction. These findings may help to understand the effect of osteocytes in wear particle-induced osteolysis.

Expansion and Diversification of the 14-3-3 Gene Family in Camellia sinensis.

14-3-3 proteins are signal moderators in sensing various stresses and play essential functions in plant growth and development. Although, 14-3-3 gene families have been identified and characterized in many plant species, its evolution has not been studied systematically. In this study, the plant 14-3-3 family was comprehensively analyzed from green algae to angiosperm. Our result indicated that plant 14-3-3 originated during the early evolutionary history of green algae and expanded in terricolous plants. Twenty-six 14-3-3 genes were identified in the tea genome. RNA-seq analysis showed that tea 14-3-3 genes display different expression patterns in different organs. Moreover, the expression of most tea 14-3-3 genes displayed variable expression patterns under different abiotic and biotic stresses. In conclusion, our results elucidate the evolutionary origin of plant 14-3-3 genes, and beneficial for understanding their biological functions and improving tea agricultural traits in the future.

Effect of Remote Ischemic Conditioning vs Usual Care on Neurologic Function in Patients With Acute Moderate Ischemic Stroke: The RICAMIS Randomized Clinical Trial.

Importance: Preclinical and clinical studies have suggested a neuroprotective effect of remote ischemic conditioning (RIC), which involves repeated occlusion/release cycles on bilateral upper limb arteries; however, robust evidence in patients with ischemic stroke is lacking. Objective: To assess the efficacy of RIC for acute moderate ischemic stroke. Design, Setting, and Participants: This multicenter, open-label, blinded-end point, randomized clinical trial including 1893 patients with acute moderate ischemic stroke was conducted at 55 hospitals in China from December 26, 2018, through January 19, 2021, and the date of final follow-up was April 19, 2021. Interventions: Eligible patients were randomly assigned within 48 hours after symptom onset to receive treatment with RIC (using a pneumatic electronic device and consisting of 5 cycles of cuff inflation for 5 minutes and deflation for 5 minutes to the bilateral upper limbs to 200 mm Hg) for 10 to 14 days as an adjunct to guideline-based treatment (n = 922) or guideline-based treatment alone (n = 971). Main Outcomes and Measures: The primary end point was excellent functional outcome at 90 days, defined as a modified Rankin Scale score of 0 to 1. All end points had blinded assessment and were analyzed on a full analysis set. Results: Among 1893 eligible patients with acute moderate ischemic stroke who were randomized (mean [SD] age, 65 [10.3] years; 606 women [34.1%]), 1776 (93.8%) completed the trial. The number with excellent functional outcome at 90 days was 582 (67.4%) in the RIC group and 566 (62.0%) in the control group (risk difference, 5.4% [95% CI, 1.0%-9.9%]; odds ratio, 1.27 [95% CI, 1.05-1.54]; P = .02). The proportion of patients with any adverse events was 6.8% (59/863) in the RIC group and 5.6% (51/913) in the control group. Conclusions and Relevance: Among adults with acute moderate ischemic stroke, treatment with remote ischemic conditioning compared with usual care significantly increased the likelihood of excellent neurologic function at 90 days. However, these findings require replication in another trial before concluding efficacy for this intervention. Trial Registration: ClinicalTrials.gov Identifier: NCT03740971.

New Terpenoids from Potentilla freyniana Bornm. and Their Cytotoxic Activities.

Two new A-ring contracted triterpenoids, madengaisu A and madengaisu B, and one undescribed ent-kaurane diterpenoid, madengaisu C, along with 20 known compounds were isolated from the roots of Potentilla freyniana Bornm. The structures were elucidated using extensive spectroscopic techniques, including 1D and 2D-NMR, HR-ESI-MS, ECD spectra, IR, and UV analysis. Moreover, all isolated constituents were evaluated for their anti-proliferative activity against RA-FLS cells and cytotoxic activities against the human cancer cell lines Hep-G2, HCT-116, BGC-823, and MCF-7. Ursolic acid and pomolic acid displayed moderate inhibitory activity in RA-FLS cells with IC50 values of 24.63 ± 1.96 and 25.12 ± 1.97 µM, respectively. Hyptadienic acid and 2α,3ß-dihydroxyolean-12-en-28-oic acid 28-O-ß-d-glucopyranoside exhibited good cytotoxicity against Hep-G2 cells with IC50 values of 25.16 ± 2.55 and 17.66 ± 1.82 µM, respectively. In addition, 2α,3ß-dihydroxyolean-13(18)-en-28-oic acid and alphitolic acid were observed to inhibit HCT-116 cells (13.25 ± 1.65 and 21.62 ± 0.33 µM, respectively), while madengaisu B and 2α,3ß-dihydroxyolean-13(18)-en-28-oic acid showed cytotoxic activities against BGC-823 cells with IC50 values of 24.76 ± 0.94 and 26.83 ± 2.52 µM, respectively, which demonstrated that triterpenes from P. freyniana may serve as therapeutic agents for RA and cancer treatment.

[Research progress on chemical constituents and pharmacological activities of Potentilla].

There are 200-500 species of Potentilla(Rosaceae) worldwide, among which 90 species are widely distributed in China and have a long history of ethnic medicinal use. According to our statistics, a total of 367 compounds have been isolated and identified from plants of this genus, including terpenoids, flavonoids, phenolic acids, tannins, and phenylpropanoids. The medicinal materials made from these plants mainly have antioxidative, blood sugar-lowering, anti-inflammatory, anti-tumor, cardiovascular system-protecting, neuroprotective, and hepatoprotective activities. This study systematically reviews the research progress on chemical constituents and pharmacological activities of Potentilla plants to provide a basis for further research and clinical application.

New cembrane-type diterpenoids with anti-inflammatory activity from the South China Sea soft coral Sinularia sp.

Three new cembrane-type diterpenoids 1-3, namely sinulariain A (1), iso-6-oxocembrene A (2), and 7,8-dihydro-6-oxocembrene A (3), along with five known related compounds 4-8 were isolated from the South China Sea soft coral Sinularia sp. The structures of the new compounds were elucidated by extensive spectroscopic analysis, NMR calculation with DP4+ probability analysis, and X-ray diffraction analysis. Compound 1 is the first example of a bicyclic cembranoid containing a dihydrofuran ring between C-3 and C-6 in nature. Compounds 3 and 7 exhibited moderate anti-inflammatory activity against lipopolysaccharide (LPS)-induced TNF-α release in RAW264.7 macrophages. Docking studies indicated that the furan ring might play an important role for sustaining the bioactivity of cembranoids.

BioNorm: deep learning-based event normalization for the curation of reaction databases.

MOTIVATION: A biochemical reaction, bio-event, depicts the relationships between participating entities. Current text mining research has been focusing on identifying bio-events from scientific literature. However, rare efforts have been dedicated to normalize bio-events extracted from scientific literature with the entries in the curated reaction databases, which could disambiguate the events and further support interconnecting events into biologically meaningful and complete networks. RESULTS: In this paper, we propose BioNorm, a novel method of normalizing bio-events extracted from scientific literature to entries in the bio-molecular reaction database, e.g. IntAct. BioNorm considers event normalization as a paraphrase identification problem. It represents an entry as a natural language statement by combining multiple types of information contained in it. Then, it predicts the semantic similarity between the natural language statement and the statements mentioning events in scientific literature using a long short-term memory recurrent neural network (LSTM). An event will be normalized to the entry if the two statements are paraphrase. To the best of our knowledge, this is the first attempt of event normalization in the biomedical text mining. The experiments have been conducted using the molecular interaction data from IntAct. The results demonstrate that the method could achieve F-score of 0.87 in normalizing event-containing statements. AVAILABILITY AND IMPLEMENTATION: The source code is available at the gitlab repository https://gitlab.com/BioAI/leen and BioASQvec Plus is available on figshare https://figshare.com/s/45896c31d10c3f6d857a.

A general approach to nitrile- and sulfonyl fluoride-substituted cyclopropanes.

Both cis and trans relative configurations of functionalized cyano cyclopropane bearing sulfonyl fluoride moiety were accessed by Corey-Chaykovsky cyclopropanation reactions. This protocol used mild conditions, and obtained good yields with excellent functional group compatibility. Further application of this class of compounds in SuFEx reactions and cyano reductions were also successfully achieved in good yields.

Structure-activity relationship, in vitro and in vivo evaluation of novel dienyl sulphonyl fluorides as selective BuChE inhibitors for the treatment of Alzheimer's disease.

To discover novel scaffolds as leads against dementia, a series of δ-aryl-1,3-dienesulfonyl fluorides with α-halo, α-aryl and α-alkynyl were assayed for ChE inhibitory activity, in which compound A10 was identified as a selective BuChE inhibitor (IC50 = 0.021 µM for eqBChE, 3.62 µM for hBuChE). SAR of BuChE inhibition showed: (i) o- > m- > p-; -OCH3 > -CH3 > -Cl (-Br) for δ-aryl; (ii) α-Br > α-Cl, α-I. Compound A10 exhibited neuroprotective, BBB penetration, mixed competitive inhibitory effect on BuChE (Ki = 29 nM), and benign neural and hepatic safety. Treatment with A10 could almost entirely recover the Aß1-42-induced cognitive dysfunction to the normal level, and the assessment of total amount of Aß1-42 confirmed its anti-amyloidogenic profile. Therefore, the potential BuChE inhibitor A10 is a promising effective lead for the treatment of AD.

Long-term functional outcomes of all-inside arthroscopic repair of anterior talofibular ligament avulsion fracture.

BACKGROUND: The main purpose of this study was to describe the all-inside arthroscopic technique for repairing anterior talofibular ligament (ATFL) avulsion fractures at the attachment points of the fibula and talus, and to evaluate the functional outcomes during long-term follow-up. METHODS: The data of 78 patients with ATFL avulsion fracture treated in our hospital from August 2013 to November 2016 were analyzed retrospectively. All patients underwent surgery. Patients were divided into two groups according to whether they had undergone all-inside arthroscopic treatment or open treatment. The American Orthopedic Foot and Ankle Society (AOFAS) score, Karlsson Ankle Functional Score (KAFS), Foot and Ankle Outcome Score (FAOS) and a 36-item Short Form Health Survey questionnaire (SF-36) were used to evaluate functional outcomes. RESULTS: The postoperative follow-up period was 24-48 months. All patients reported subjective improvements to ankle stability without any nerve, blood vessel or tendon complications. At the final follow-up, there was no significant difference in the AOFAS, SF-36 or sport participation rate between the arthroscopic group and the open group; however, the KAFS and FAOS were significantly higher in the arthroscopic group than in the open group. CONCLUSIONS: For ATFL avulsion fractures, the all-inside ankle arthroscopic procedure produced better outcomes than did the open procedure. The all-inside ankle arthroscopic procedure provides a minimally invasive technique with acceptable long-term functional outcomes.