Development of new techniques and clinical applications of liquid biopsy in lung cancer management.

Lung cancer is an exceedingly malignant tumor reported as having the highest morbidity and mortality of any cancer worldwide, thus posing a great threat to global health. Despite the growing demand for precision medicine, current methods for early clinical detection, treatment and prognosis monitoring in lung cancer are hampered by certain bottlenecks. Studies have found that during the formation and development of a tumor, molecular substances carrying tumor-related genetic information can be released into body fluids. Liquid biopsy (LB), a method for detecting these tumor-related markers in body fluids, maybe a way to make progress in these bottlenecks. In recent years, LB technology has undergone rapid advancements. Therefore, this review will provide information on technical updates to LB and its potential clinical applications, evaluate its effectiveness for specific applications, discuss the existing limitations of LB, and present a look forward to possible future clinical applications. Specifically, this paper will introduce technical updates from the prospectives of engineering breakthroughs in the detection of membrane-based LB biomarkers and other improvements in sequencing technology. Additionally, it will summarize the latest applications of liquid biopsy for the early detection, diagnosis, treatment, and prognosis of lung cancer. We will present the interconnectedness of clinical and laboratory issues and the interplay of technology and application in LB today.

Towards the molecular era of discriminating multiple lung cancers.

In the era of histopathology-based diagnosis, the discrimination between multiple lung cancers (MLCs) poses significant uncertainties and has thus become a clinical dilemma. However, recent significant advances and increased application of molecular technologies in clonal relatedness assessment have led to more precision in distinguishing between multiple primary lung cancers (MPLCs) and intrapulmonary metastasis (IPMs). This review summarizes recent advances in the molecular identification of MLCs and compares various methods based on somatic mutations, chromosome alterations, microRNAs, and tumor microenvironment markers. The paper also discusses current challenges at the forefront of genomics-based discrimination, including the selection of detection technology, application of next-generation sequencing, and intratumoral heterogeneity (ITH). In summary, this paper highlights an entrance into the primary stage of molecule-based diagnostics.

Case Report: A Novel CACNA1A Mutation Caused Flunarizine-Responsive Type 2 Episodic Ataxia and Hemiplegic Migraine With Abnormal MRI of Cerebral White Matter.

Episodic ataxia type 2 (EA2) is one autosomal-dominant neurological disorder characterized by debilitating attacks of ataxia. It is mainly caused by loss-of-function mutations of the CACNA1A gene, which encodes the pore-forming α1A subunit of Cav2.1 (P/Q type voltage-gated calcium channel). Sporadic hemiplegic migraine (SHM) is another rare disease involving CACNA1A variants, which seldom coexists with EA2. Here we report a novel pathogenic mutation in CACNA1A (c.3836dupA, exon 23, p.Y1279X) of a 16-year-old female, who complained about paroxysmal dizziness, headache, and unsteady gait. Her brain MRI revealed a slightly atrophic cerebellum and numerous asymptomatic hyperintense lesions of the cerebral white matter. The diagnosis of EA2 combined with SHM was made. Administration of 5-mg flunarizine once daily at night effectively reduced the attacks and attenuated her symptoms for a month.

Efficacy and Safety of Tacrolimus Therapy for a Single Chinese Cohort With Very-Late-Onset Myasthenia Gravis.

Background and Purpose: Previous studies have found tacrolimus to be a favorable drug for treating different types of myasthenia gravis (MG), but few have focused on very-late-onset MG (VLOMG). This study evaluated the efficacy and safety of tacrolimus for VLOMG therapy. Methods: This was a retrospective single-center cohort study of 70 patients with VLOMG (onset ≥65 years) who visited Peking University First Hospital in 2019. Participants were divided into the tacrolimus (Tac) group and the control group based on tacrolimus usage. We further divided the Tac group into patients treated without corticosteroids and with corticosteroids. Sociodemographic features, clinical profiles, and outcomes were compared between different therapies and further analyzed by multivariate regression. Details of tacrolimus treatment, comorbidities, and adverse drug reactions (ADRs) were described. Results: Among 70 patients, the median (interquartile range) age at onset was 71 (68-77) years, and the follow-up duration was 27 (27-29) months. Most patients were types I (28%) and III (40%) according to the MG Foundation of America (MGFA) classification. In the Tac group, tacrolimus treatment was maintained for 36 (27-38) months. The dosage at the final evaluation was 1.0 (1.0-1.75) mg/day, and the last blood concentration test was 4.25 (2.85-5.7) ng/ml. A total of 43% reached remission, and 37% improved based on MGFA postintervention status (MGFA-PIS). For the 9 patients, newly diagnosed at enrollment within this group, MG activities of daily living (MG-ADL) decreased significantly from 3 (2-5) to 2 (1-2) (p = 0.041). Regarding the 13 patients, coadministering Wuzhi capsules the tacrolimus concentration increased from 2.75 (1.4-3.8) ng/ml to 5.95 (5.1-7.0) ng/ml (p = 0.012). No significant differences in outcomes were observed between tacrolimus treatment without and with corticosteroids or between the Tac group and the control group. A total of 93% had at least one comorbidity. ADRs related to tacrolimus emerged in 25% (9/36) of patients, most of which were not serious and reversible. Conclusions: Tacrolimus is effective and safe in treating VLOMG. Tacrolimus monotherapy without corticosteroids can be used as an initial and maintenance treatment for VLOMG. Wuzhi capsules work well in elevating tacrolimus concentrations in this population.

The Effect of the NFκB-USP9X-Cx43 Axis on the Dynamic Balance of Bone Formation/Degradation during Ossification of the Posterior Longitudinal Ligament of the Cervical Spine.

Connexin 43- (Cx43-) mediated nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling has been found involved in the ossification of the posterior longitudinal ligament (OPLL). However, the underlying mechanism how OPLL is regulated has not been elucidated. In the present study, primary ligament fibroblast were isolated; immunoprecipitation (IP) and liquid chromatography-mass spectrometry (LC-MS) assays were applied to identify potential binding proteins of Cx43. Protein interaction was then confirmed by co-IP assay. Alkaline phosphatase (ALP) activity and alizarin red staining were used to evaluate ossification. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were employed to assess the binding between NF-κB p65 and target gene. Lipoxygenase inhibitor (5,8,11-eicosatriynoic acid, EPA) was applied to induce endoplasmic reticulum (ER) stress, and 4-phenylbutyrate (4-PBA) was used as an ER-stress inhibitor. Expression of USP9X, Cx43, and nuclei p65 in ligaments from patients and controls was detected by Western blotting. The results showed that ubiquitin-specific protease 9 X-chromosome (USP9X), a deubiquitylating enzyme, was a candidate of Cx43 binding proteins, and USP9X inhibited Cx43 ubiquitination. In vitro experiments showed that USP9X promoted ossification of primary ligament fibroblasts and nuclear translocation of NF-κB p65 by regulating Cx43 expression. Moreover, NF-κB can bind to the USP9X promoter to promote its transcription. When ER stress was inhibited by 4-PBA, USP9X levels, NF-κB nuclei translocation, and ALP activity were decreased. Reverse results were obtained when ER stress was induced by EPA. PDTC, an NF-κB inhibitor, could abolish the effects of EPA. Furthermore, USP9X, Cx43, and nuclei p65 were significantly upregulated in ligaments from OPLL patients than non-OPLL controls. USP9X was positively correlated with CX43 and nuclei p65 in OPLL samples. Overall, the findings suggest that the ER stress-NFκB-USP9X-Cx43 signaling pathway plays an important role in OPLL progression.

Urodynamic evaluation of bladder function in patients with urinary incontinence secondary to congenital tethered cord syndrome after homogeneous spinal-shortening axial decompression procedure.

OBJECTIVE: To evaluate the effect of homogeneous spinal-shortening axial decompression procedure (HSAD) on bladder function in patients with spina bifida tethered syndrome. METHODS AND MATERIALS: Patients with tethered spinal cord syndrome were collected prospectively, and all patients were treated with lumbar HSAD. Patients' urodynamic evaluation mainly included detrusor function, sphincter function, sphincter coordination (Ig TLR, ratio of tension and loose of urethral sphincter), and bladder compliance. Meanwhile, all patients were followed up with ICI-Q-SF, SF-12, and Rantala scores. RESULTS: Twenty-four patients were included, with the average age of 27 ± 16 years. At the final follow-up, patients' detrusor function, sphincter function, sphincter coordination, and bladder compliance, were all improved dramatically (all P < 0.01). The preoperative SF-12 score, ICQ, and Rantala score were [52.16 ± 5.64, 14.11 ± 5.25, 7.84 ± 4.87], whereas the postoperative mean was [33.53 ± 3.53, 9.05 ± 4.89, 15 ± 3.77] (P < 0.01, respectively). According to objective evaluation, 16.7% of them recovered to normal. According to the subjective evaluation, 25% of the patients returned to normal. Only one patient (4.2%) deteriorated. Limitations include none-randomized controlled design and limited patient samples. CONCLUSIONS: The HSAD can significantly restore the bladder function in patients with long-term urinary incontinence.

An open-label randomized multi-Centre study to evaluate anterior controllable Antedisplacement and fusion versus posterior Laminoplasty in patients with cervical ossification of the posterior longitudinal ligament: study design and analysis plan (STAR).

BACKGROUND: In treating patients with cervical ossification of the posterior longitudinal ligament (COPLL), a novel surgery technique - anterior controllable antedisplacement and fusion (ACAF) suggested promising clinical benefits in recent exploratory studies. METHODS: This is a multicentre, randomized, open-label, parallel-group, active controlled trial that will compare the clinical benefits of ACAF versus conventional posterior laminoplasty (LAMP) in severe COPLL patients. A total of 164 patients will be enrolled and randomized in a 1:1 ratio to either ACAF or LAMP group. The primary efficacy measure is cervical- Japanese Orthopaedic Association (C-JOA) recovery rate at 12 months post operation, which is to be derived by Hirabayashi's method from JOA data (range, 0 [worst] to 17 [normal condition]). Other important secondary efficacy endpoints include visual analogue scale (VAS) pain score (range, 0 [no pain] to 10 [most severe]), 10-item neck disability index (NDI, a total range of 0 to 50 points, the highest index the worst) and 6-level Nurick disability grade (range, 0 [mild] to 5 [severe]). Safety endpoints including adverse events, perioperative complications, and adverse events of special interest will also be assessed in this study. Full analysis set for baseline and efficacy data analyses according to the intention-to-treat principle will be established as the primary analysis population. Analysis of covariance (ANCOVA) will be used to analyze the C-JOA recovery rate, with random stratification factors (if appropriate) and the treatment group as fixed factors, and the baseline level of C-JOA score as covariate. DISCUSSION: This study is designed to demonstrate the clinical benefits of ACAF as compared to conventional LAMP in COPLL patients. It will provide clinical evidence that the novel surgery technique - ACAF might be more favorable in treating patients with severe cervical ossification of the posterior longitudinal ligament. (Words: 290). TRIAL REGISTRATION: ClinicalTrials.gov number, NCT04968028 .

Human umbilical cord mesenchymal stem cells deliver exogenous miR-26a-5p via exosomes to inhibit nucleus pulposus cell pyroptosis through METTL14/NLRP3.

BACKGROUND: Intervertebral disc degeneration (IVDD) is the breakdown of the discs supporting the vertebrae. It is one of the most frequent causes of back pain worldwide. Currently, the clinical interventions for IVDD are mainly focused on symptom releases. Thus, new therapeutic options are needed. METHODS: Nucleus pulposus (NP) samples were obtained from 20 patients experiencing IVDD and 10 healthy volunteers compared for mRNA N6-methyladenosine (m6A) mRNA modification as well as methyltransferase (METT) like METTL3, METTL14, and Wilms' tumor 1-associated protein mRNA and protein abundance following exosomes exposure from mesenchymal stem cells. In addition, microRNA expressions were also compared. The correlation between the NLR family pyrin domain containing 3 (NLRP3) and METTL14 was measured by luciferase reporter assay. Cytokines were evaluated using an enzyme-linked immunosorbent assay. METTL14, NLRP3, and insulin-like growth factor 2 mRNA-binding protein 2 mRNAs were measured via a quantitative reverse transcription-polymerase chain reaction. Protein was assayed using western blots. Cell death was assessed by propidium iodide staining, lactate dehydrogenase release, gasdermin-N domain abundance, and caspase-1 activation. RESULTS: The human umbilical cord mesenchymal stem cell (hucMSC) exosomes were found to effectively improve the viability of NP cells and protect them from pyroptosis through targeting METTL14, with a methyltransferase catalyzing m6A modification. METTL14 was highly present in NP cells from IVDD patients, which stabilize NLRP3 mRNA in an IGFBP2-dependent manner. The elevated NLRP3 levels result in the increase of interleukin 1ß (IL-1ß) and IL-18 levels and trigger pyroptotic NP cell death. Such pathogenic axis could be blocked by hucMSC exosomes, which directly degrade METTL14 through exosomal miR-26a-5p. CONCLUSIONS: The results of the current study revealed the beneficial effects of hucMSC exosomes on NP cells and determined a potential mechanism inducing IVDD.

METTL3 Regulates Ossification of the Posterior Longitudinal Ligament via the lncRNA XIST/miR-302a-3p/USP8 Axis.

The prevalence of ossification of the posterior longitudinal ligament (OPLL) is increasing, and currently there is no effective medical treatment for OPLL. Methyltransferase like 3 (METTL3), one of the components of the N 6-methyladenosine (m6A) methyltransferase complex, regulates gene expression via modification of mRNA. Although METTL3 has been implicated in a variety of diseases, its role in OPLL remains to be elucidated. Primary ligament fibroblasts were used in this study. To investigate the role of METTL3 in OPLL, METTL3 was silenced or overexpressed. m6A RNA methylation was measured by commercially available kits. Luciferase reporter assay was performed to investigate the binding of miR-302a-3p and METTL3, and the binding of miR-302a-3p and USP8. Quantitative RT-PCR and western blots were used to evaluate mRNA and protein expression, respectively. OPLL increases METTL3 and its m6A modification. Overexpressing METTL3 significantly promoted osteogenic differentiation of primary ligament fibroblasts. Mechanism study showed that METTL3 increased m6A methylation of long non-coding RNA (lncRNA) X-inactive specific transcript (XIST). Further study showed that lncRNA XIST regulates osteogenic differentiation of primary ligament fibroblasts via miR-302a-3p, which targets ubiquitin-specific protease 8 (USP8). METTL3 enhanced osteogenic differentiation of primary ligament fibroblasts via the lncRNA XIST/miR-302a-3p/USP8 axis. The findings highlight the importance of METTL3-mediated m6A methylation of XIST in OPLL and provide new insights into therapeutic strategies for OPLL.

Non-coding RNAs in ossification of spinal ligament.

PURPOSE: Ossification of the spinal ligament (OSL) is a disease characterized by progressive ectopic ossification or calcification in the tissues of spinal ligament. The molecular pathogenesis of OSL has not been clearly elucidated. Recently, ncRNAs was found to functionally participate in OSL development. This review summarized current knowledge regarding the deregulation and function of ncRNAs in OSL METHODS: Relevant studies on deregulation and function of ncRNAs in OSL were retrieved from the PubMed databases. Then, studies were manually selected for inclusion based on predefined criteria. RESULT: 14 studies were reviewed, with 4 studies about high throughput sequencing and microarray of ncRNAs, 8 studies relevant to the function of ncRNAs and 2 studies regarding the ncRNAs as the biomarker of OSL. CONCLUSION: ncRNA play a vital role in the ossification of spinal ligament fibrocyte, including cell osteogenesis and inflammation. ncRNAs also have potential clinical utilities as therapeutic targets, risk predication and early detection in the management of OSL. LEVEL OF EVIDENCE I: Diagnostic: individual cross-sectional studies with the consistently applied reference standard and blinding.

Single-water-dipole-layer-driven Reversible Charge Order Transition in 1T-TaS2.

Water-solid interactions are crucial for many fundamental phenomena and technological processes. Here, we report a scanning tunneling microscopy study about the charge density wave (CDW) transition in 1T-TaS2 driven by a single water dipole layer. At low temperature, pristine 1T-TaS2 is a prototypical CDW compound with 13 × 13 charge order. After growing a highly ordered water adlayer, a new charge order with 3 × 3 periodicity emerges on water-covered 1T-TaS2. After water desorption, the entire 1T-TaS2 surface appears as localized 13 × 13 CDW domains that are separated by residual-water-cluster-pinned CDW domain walls. First-principles calculations show that the electric dipole moments in the water adlayer attract electrons to the top layer of 1T-TaS2, which shifts the phonon softening mode and induces the 13 × 13 to 3 × 3 charge order transition. Our results pave the way for creating new collective quantum states of matter with a molecular dipole layer.

Reliability and validity of multi-shot DWI in diagnosis of cervical spondylotic myelopathy: a study based on 3-T MRI.

OBJECTIVE: To investigate whether multi-shot diffusion-weighted imaging (ms-DWI) could be applied in diagnosis and quantitative evaluation of cervical spondylotic myelopathy (CSM). METHODS: Thirty-three normal volunteers and 78 patients with CSM were included in this study. The apparent diffusion coefficient (ADC) values were measured at C2-C7 levels on sagittal section ADC map. The intraclass correlation coefficient (ICC) and Bland and Altman plot and Spearman coefficient were used to quantify the reproducibility of test and retest and inter-rater reliability. Pearson correlations were calculated to compare lADC and rADC versus mJOA and NDI scores. Receiver operating characteristic curve and the area under the curve (AUC) were applied to evaluate the diagnostic reliability. RESULTS: There was no statistically significant difference between ADC values obtained from normal volunteers at C2-C7 levels (P < 0.05). The ICC and spearman coefficient of lADC and rADC indicated excellent test-retest and inter-rater reliability. The mean lADC and rADC were significantly higher from patients than that from volunteers (all P < 0.01). The lADC had moderate to good correlations with mJOA and NDI (all P < 0.0001). Moreover, rADC had good to excellent correlations with mJOA and NDI (all P < 0.0001). Comparing AUCs, rADC was significantly superior in diagnosis which participants were CSM than lADC (P = 0.0118). CONCLUSION: The ms-DWI could be applied in diagnosis and quantitive assessment of CSM according to lADC and rADC. A new parameter, rADC, could be served as a diagnostic indice for CSM, which may quantitively reflect the severity of CSM. These slides can be retrieved under Electronic Supplementary Material.

Site-Specific Photo-Crosslinking Proteomics Reveal Regulation of IFITM3 Trafficking and Turnover by VCP/p97 ATPase.

Interferon-induced transmembrane protein 3 (IFITM3) is a key interferon effector that broadly prevents infection by diverse viruses. However, the cellular factors that control IFITM3 homeostasis and antiviral activity have not been fully elucidated. Using site-specific photo-crosslinking and quantitative proteomic analysis, here we present the identification and functional characterization of VCP/p97 AAA-ATPase as a primary interaction partner of IFITM3. We show that IFITM3 ubiquitination at lysine 24 is crucial for VCP binding, trafficking, turnover, and engagement with incoming virus particles. Consistently, pharmacological inhibition of VCP/p97 ATPase activity leads to defective IFITM3 lysosomal sorting, turnover, and co-trafficking with virus particles. Our results showcase the utility of site-specific protein photo-crosslinking in mammalian cells and reveal VCP/p97 as a key cellular factor involved in IFITM3 trafficking and homeostasis.

Comparison of Anterior Controllable Antedisplacement and Fusion With Posterior Laminoplasty in the Treatment of Multilevel Cervical Ossification of the Posterior Longitudinal Ligament: A Prospective, Randomized, and Control Study With at Least 1-Year Follow Up.

STUDY DESIGN: A prospective, randomized, controlled study. OBJECTIVE: To compare anterior controllable antidisplacement and fusion (ACAF) with laminoplasty in the treatment of multilevel ossification of the posterior longitudinal ligament (OPLL), and evaluate the efficacy and safety of this procedure. SUMMARY OF BACKGROUND DATA: The optimal approach for the treatment of OPLL still remains controversial. Both anterior and posterior approaches have their advantages and disadvantages. METHODS: Between September 2016 and April 2018, a total of 80 patients with multilevel OPLL were randomized in a 1:1 ratio to ACAF group and laminoplasty group. All patients were followed up at least 1 year. Clinical and radiological results were compared between ACAF group and laminoplasty group. RESULTS: ACAF took a longer operation time. C5 palsy and axial pain occurred more commonly in laminoplasty group, whereas dysphagia and hoarseness appeared easily in ACAF group. At 1-year follow-up, the final Japanese Orthopedic Association (JOA) score and recovery rate were significant higher in ACAF group than those in laminoplasty group, when occupying rate (OR) was not less than 60%, or K-line was negative. ACAF was also good at preservation of cervical lordosis and sagittal balance, but range of movement of cervical spine in both groups decreased significantly. CONCLUSION: Generally speaking, ACAF is a safe and effective alternative for multilevel OPLL. Compared with laminoplasty, ACAF is more effective in the cases when OR is not less than 60%, or K-line is negative. LEVEL OF EVIDENCE: 2.

Long non-coding RNA MALAT1 functions as miR-1 sponge to regulate Connexin 43-mediated ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) is the major cause for several deteriorate bone and joint diseases. Its development is a highly organized dynamic process as modulated by various physiological and pathophysiological factors. Both long non-coding RNAs (lncRNAs) and small non-coding RNAs (miRNAs) have been postulated to involve into almost all the biological conditions. Here, we applied high through-put transcriptome screening to unveil lncRNAs highly regulated under OPLL condition. siRNA assay in combination with western blot and quantitative PCR deciphered the lncRNA and miRNA functions in OPLL and their underlying mechanism. Here we identified an lncRNA, named Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) engaged into the development of OPLL by indirectly targeting Connexin 43 (Cx43) gene. As previously reported, Cx43 is one of the main proteins contributing to OPLL partially through enhancing inflammatory signaling. On top of that, we provided another regulatory layer that MALAT1 served as the upstream effector governing the transcription of Cx43 gene. Perturbation of MALAT1 significantly inhibited Cx43 expression, inflammation, and osteogenesis. Mechanistically, in silico analysis and experimental validation both confirmed that microRNA-1 (miR-1) was the mediator connecting MALAT1-Cx43 axis: overexpression of miR-1 diminished Cx43 expression and OPLL process; meanwhile, MALAT1 acted as miR-1 sponge to inhibit its suppressive transcription effect on downstream ossification related genes. Knock-down of MALAT1 released sequestered miR-1, which repressed Cx43 expression and associated OPLL. Likewise, induced OPLL caused by overexpression of MALAT1 can be ameliorated by enhanced miR-1 function, knock-down of Cx43 or inhibition of inflammation. More importantly, further validation using patient ligament samples from non-OPLL and OPLL individuals identified MALAT1-miR-1-Cx43 regulatory axis. Collectively, we found a novel mechanism through lncRNA-miRNA interaction that provides more insights into understanding the development of OPLL.

How to Avoid Postoperative Remaining Ossification Mass in Anterior Controllable Antedisplacement and Fusion Surgery.

OBJECTIVES: To retrospectively review the cases with ossification of the posterior longitudinal ligament (OPLL) treated with anterior controllable antedisplacement and fusion (ACAF). Patients with postoperative remaining ossification mass (PROM) are analyzed to figure out the causes and preventions of this problem. METHODS: A total of 115 patients were included. PROM was identified as remaining OPLL existed in the spinal canal other than included in the vertebral-OPLL complex on postoperative computed tomography. The Japanese Orthopaedic Association scoring system was used to evaluate the neurologic status. Surgery-related complications such as cerebrospinal fluid (CSF) leakage and spinal cord or nerve injury were all recorded. The patients with the PROM group and those without the PROM group were compared. RESULTS: There were 14 patients with wide-base OPLL (12.2%) and 10 patients (8.7%) with PROM among the 115 patients with OPLL. The 10 patients with PROM were all with wide-base OPLL. The average improvement rate of Japanese Orthopaedic Association score in patients without PROM was significantly larger than that in patients with PROM (69.5 ± 22.6% vs. 36.7 ± 22.0, P < 0.01). Incidence rate of postoperative CSF leakage and neural deterioration were significantly higher in patients with PROM than that in patients without PROM (CSF leakage, 40.0% vs. 5.9%; neural deterioration, 50.0% vs. 3.0%). No other complications were observed. CONCLUSIONS: The occurrence of PROM might cause complications and poor neural function recovery in patients treated with ACAF. Surgical techniques should be noted to avoid PROM in ACAF surgery.

Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation.

Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferon-induced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.

Turning the spotlight on protein-lipid interactions in cells.

Protein function is largely dependent on coordinated and dynamic interactions of the protein with biomolecules including other proteins, nucleic acids and lipids. Although powerful methods for global profiling of protein-protein and protein-nucleic acid interactions are available, proteome-wide mapping of protein-lipid interactions is still challenging and rarely performed. The emergence of bifunctional lipid probes with photoactivatable and clickable groups offers new chemical tools for globally profiling protein-lipid interactions under cellular contexts. In this review, we summarize recent advances in the development of bifunctional lipid probes for studying protein-lipid interactions. We also highlight how in vivo photocrosslinking reactions contribute to the characterization of lipid-binding proteins and lipidation-mediated protein-protein interactions.

Single amino acid alteration between valine and isoleucine determines the distinct pyrabactin selectivity by PYL1 and PYL2.

Abscisic acid (ABA) is one of the most important phytohormones in plant. PYL proteins were identified to be ABA receptors in Arabidopsis thaliana. Despite the remarkably high degree of sequence similarity, PYL1 and PYL2 exhibit distinct responses toward pyrabactin, an ABA agonist. PYL1 inhibits protein phosphatase type 2C upon binding of pyrabactin. In contrast, PYL2 appears relatively insensitive to this compound. The crystal structure of pyrabactin-bound PYL1 revealed that most of the PYL1 residues involved in pyrabactin binding are conserved, hence failing to explain the selectivity of pyrabactin for PYL1 over PYL2. To understand the molecular basis of pyrabactin selectivity, we determined the crystal structure of PYL2 in complex with pyrabactin at 1.64 A resolution. Structural comparison and biochemical analyses demonstrated that one single amino acid alteration between a corresponding valine and isoleucine determines the distinct pyrabactin selectivity by PYL1 and PYL2. These characterizations provide an important clue to dissecting the redundancy of PYL proteins.

Functional mechanism of the abscisic acid agonist pyrabactin.

Pyrabactin is a synthetic abscisic acid (ABA) agonist that selectively inhibits seed germination. The use of pyrabactin was pivotal in the identification of the PYR1/PYL/RCAR family (PYL) of proteins as the ABA receptor. Although they both act through PYL proteins, pyrabactin and ABA share no apparent chemical or structural similarity. It remains unclear how pyrabactin functions as an ABA agonist. Here, we report the crystal structure of pyrabactin in complex with PYL1 at 2.4 A resolution. Structural and biochemical analyses revealed that recognition of pyrabactin by the pocket residues precedes the closure of switch loop CL2. Structural comparison between pyrabactin- and ABA-bound PYL1 reveals a general principle in the arrangements of function groups of the two distinct ligands. The study provides a framework for the development of novel ABA agonists that may have applicable potentials in agriculture.