The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.

Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.

Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts.

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.

DFT-Assisted Investigation of the Electric Field and Charge Density Distribution of Pristine and Defective 2D WSe2 by Differential Phase Contrast Imaging.

Most properties of solid materials are defined by their internal electric field and charge density distributions which so far are difficult to measure with high spatial resolution. Especially for 2D materials, the atomic electric fields influence the optoelectronic properties. In this study, the atomic-scale electric field and charge density distribution of WSe2 bi- and trilayers are revealed using an emerging microscopy technique, differential phase contrast (DPC) imaging in scanning transmission electron microscopy (STEM). For pristine material, a higher positive charge density located at the selenium atomic columns compared to the tungsten atomic columns is obtained and tentatively explained by a coherent scattering effect. Furthermore, the change in the electric field distribution induced by a missing selenium atomic column is investigated. A characteristic electric field distribution in the vicinity of the defect with locally reduced magnitudes compared to the pristine lattice is observed. This effect is accompanied by a considerable inward relaxation of the surrounding lattice, which according to first principles DFT calculation is fully compatible with a missing column of Se atoms. This shows that DPC imaging, as an electric field sensitive technique, provides additional and remarkable information to the otherwise only structural analysis obtained with conventional STEM imaging.

Differential phase contrast (DPC) mapping electric fields: Optimising experimental conditions.

DPC in Scanning Transmission Electron Microscopy (STEM) is a valuable method for mapping the electric fields in semiconductor materials. However, optimising the experimental conditions can be challenging. In this paper, we test and compare critical experimental parameters, including the convergence angle, camera length, acceleration voltage, sample configuration, and orientation using a four-quadrant segmented detector and a Si specimen containing layers of different As concentrations. The DPC measurements show a roughly linear correlation with the estimated electric fields, until the field gets close to the detection limitation, which is ∼0.5 mV/nm with a sample thickness of ∼145 nm. These results can help inform which technique to use for different user cases: When the electric field at a planar junction is above ∼0.5 mV/nm, DPC with a segmented detector is practical for electric field mapping. With a planar junction, the DPC signal-to-noise ratio can be increased by increasing the specimen thickness. However, for semiconductor devices with electric fields smaller than ∼0.5 mV/nm, or for devices containing curved junctions, DPC is unreliable and techniques with higher sensitivity will need to be explored, such as 4D STEM using a pixelated detector.

ADAM10 and ADAM17 promote SARS-CoV-2 cell entry and spike protein-mediated lung cell fusion.

The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.

Exosomes derived from mouse vibrissa dermal papilla cells promote hair follicle regeneration during wound healing by activating Wnt/ß-catenin signaling pathway.

Hair follicle (HF) regeneration during wound healing continues to present a significant clinical challenge. Dermal papilla cell-derived exosomes (DPC-Exos) hold immense potential for inducing HF neogenesis. However, the accurate role and underlying mechanisms of DPC-Exos in HF regeneration in wound healing remain to be fully explained. This study, represents the first analysis into the effects of DPC-Exos on fibroblasts during wound healing. Our findings demonstrated that DPC-Exos could stimulate the proliferation and migration of fibroblasts, more importantly, enhance the hair-inducing capacity of fibroblasts. Fibroblasts treated with DPC-Exos were capable of inducing HF neogenesis in nude mice when combined with neonatal mice epidermal cells. In addition, DPC-Exos accelerated wound re-epithelialization and promoted HF regeneration during the healing process. Treatment with DPC-Exos led to increased expression levels of the Wnt pathway transcription factors ß-catenin and Lef1 in both fibroblasts and the dermis of skin wounds. Specifically, the application of a Wnt pathway inhibitor reduced the effects of DPC-Exos on fibroblasts and wound healing. Accordingly, these results offer evidence that DPC-Exos promote HF regeneration during wound healing by enhancing the hair-inducing capacity of fibroblasts and activating the Wnt/ß-catenin signaling pathway. This suggests that DPC-Exos may represent a promising therapeutic strategy for achieving regenerative wound healing.

Fast Automatic Fuzzy C-Means Knitting Pattern Color-Separation Algorithm Based on Superpixels.

Patterns entered into knitting CAD have thousands or tens of thousands of different colors, which need to be merged by color-separation algorithms. However, for degraded patterns, the current color-separation algorithms cannot achieve the desired results, and the clustering quantity parameter needs to be managed manually. In this paper, we propose a fast and automatic FCM color-separation algorithm based on superpixels, which first uses the Real-ESRGAN blind super-resolution network to clarify the degraded patterns and obtain high-resolution images with clear boundaries. Then, it uses the improved MMGR-WT superpixel algorithm to pre-separate the high-resolution images and obtain superpixel images with smooth and accurate edges. Subsequently, the number of superpixel clusters is automatically calculated by the improved density peak clustering (DPC) algorithm. Finally, the superpixels are clustered using fast fuzzy c-means (FCM) based on a color histogram. The experimental results show that not only is the algorithm able to automatically determine the number of colors in the pattern and achieve the accurate color separation of degraded patterns, but it also has lower running time. The color-separation results for 30 degraded patterns show that the segmentation accuracy of the color-separation algorithm proposed in this paper reaches 95.78%.

Estimated number of spine surgeries and related deaths in Japan from 2014 to 2020.

BACKGROUND: The total number of spine surgeries per year and their related deaths in Japan has not been adequately estimated in the literature. METHODS: We retrospectively reviewed the National Database of Health Insurance Claims and Specific Health Checkups of Japan (NDB) between April 2014 to March 2021, which covers 99.9 % of health insurance claim receipts by general practitioners. The annual number of surgeries was counted using K codes, a procedure classification unique to Japan, and classified into the following six categories; percutaneous vertebroplasty, endoscopic surgery, open discectomy, laminoplasty/laminectomy, instrumentation surgery, and others. The data distribution was also summarized by sex and age. Additionally, by reviewing DPC database-related papers for evaluation of the mortality rate after spine surgery in Japan, the number of spine surgery-related deaths was calculated. RESULTS: The NDB showed that the number of spine surgeries analyzed in this study increased from 170,081 in 2014 to 193,903 in 2019, with a slight decrease in 2020. The ratio of instrumentation surgery increased from 33.0 % in 2014 to 37.9 % in 2020. The rate of patients aged 75 or older increased 31.6 % to 39.6 %. Combining these findings with DPC data showing a mortality rate of 0.1 % to 0.4 % revealed that the estimated number of deaths related to spine surgery in Japan ranged from 200 to 800 per year. CONCLUSIONS: Approximately 200,000 spine surgeries and 200 to 800 spine surgery-related inpatient deaths were estimated to have occurred in Japan around 2020.

Patient characteristics, diagnostic testing, and initial treatment profiles of patients with connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH) using a Japanese claims database.

OBJECTIVES: To investigate the diagnostic and therapeutic landscape for patients with connective tissue disease (CTD) and CTD-associated pulmonary arterial hypertension (CTD-PAH) in acute-care general hospitals in Japan. METHODS: We conducted a retrospective cohort study by analysing the Medical Data Vision (MDV) database from April 2008 and September 2020. CTD patients who prescribed immunosuppressants were included in cohort 1, and CTD-PAH patients extracted from cohort 1 were included in cohort 2. Patient characteristics, diagnostic screening frequencies for PAH, and initial PAH-specific treatment patterns were assessed. RESULTS: Overall, 16648 patients with CTD and 81 patients with CTD-PAH were included in cohorts 1 and 2, respectively. The frequencies of screening tests for PAH, including brain natriuretic peptide (BNP), transthoracic echocardiogram (TTE), and diffusing capacity of the lungs for carbon monoxide (DLCO), among CTD patients were 0.7, 0.3, and 0.1 tests/person-year, respectively. The most common initial PAH-specific treatment therapy was monotherapy (87.7%), followed by dual therapy (7.4%), and triple therapy (2.5%). CONCLUSION: This is the first study to describe the patient flow from PAH diagnosis to initial PAH-specific treatment for real-world patients who were followed regularly due to CTD in Japanese clinical practice.

The SOS response-associated peptidase (SRAP) domain of YedK catalyzes ring opening of abasic sites and reversal of its DNA-protein cross-link.

Apurinic/apyrimidinic (AP, or abasic) sites in DNA are one of the most common forms of DNA damage. AP sites are reactive and form cross-links to both proteins and DNA, are prone to strand breakage, and inhibit DNA replication and transcription. The replication-associated AP site repair protein HMCES protects cells from strand breaks, inhibits mutagenic translesion synthesis, and participates in repair of interstrand DNA cross-links derived from AP sites by forming a stable thiazolidine DNA-protein cross-link (DPC) to AP sites in single-stranded DNA (ssDNA). Despite the importance of HMCES to genome maintenance and the evolutionary conservation of its catalytic SRAP (SOS Response Associated Peptidase) domain, the enzymatic mechanisms of DPC formation and resolution are unknown. Using the bacterial homolog YedK, we show that the SRAP domain catalyzes conversion of the AP site to its reactive, ring-opened aldehyde form, and we provide structural evidence for the Schiff base intermediate that forms prior to the more stable thiazolidine. We also report two new activities, whereby SRAP reacts with polyunsaturated aldehydes at DNA 3'-ends generated by bifunctional DNA glycosylases and catalyzes direct reversal of the DPC to regenerate the AP site, the latter of which we observe in both YedK and HMCES-SRAP proteins. Taken together, this work provides insights into possible mechanisms by which HMCES DPCs are resolved in cells.

Differential phase contrast for quantitative imaging and spectro-microscopy at a nanoprobe beamline.

The interaction of a focused X-ray beam with a sample in a scanning probe experiment can provide a variety of information about the interaction volume. In many scanning probe experiments X-ray fluorescence (XRF) is supplemented with measurements of the transmitted or scattered intensity using a pixelated detector. The automated extraction of different signals from an area pixelated detector is described, in particular the methodology for extracting differential phase contrast (DPC) is demonstrated and different processing methods are compared across a range of samples. The phase shift of the transmitted X-ray beam by the sample, extracted from DPC, is also compared with ptychography measurements to provide a qualitative and quantitative comparison. While ptychography produces a superior image, DPC can offer a simple, flexible method for phase contrast imaging which can provide fast results and feedback during an experiment; furthermore, for many science problems, such as registration of XRF in a lighter matrix, DPC can provide sufficient information to meet the experimental aims. As the DPC technique is a quantitative measurement, it can be expanded to spectroscopic studies and a demonstration of DPC for spectro-microscopy measurements is presented. Where ptychography can separate the absorption and phase shifts by the sample, quantitative interpretation of a DPC image or spectro-microscopy signal can only be performed directly when absorption is negligible or where the absorption contribution is known and the contributions can be fitted.

Continuous near-infrared reflectance spectroscopy monitoring to guide distal perfusion can minimize limb ischemia surgery for patients requiring femoral venoarterial extracorporeal life support.

BACKGROUND: Patients requiring femoral venoarterial (VA) extracorporeal life support (ECLS) are at risk of distal lower limb hypoperfusion and ischemia of the cannulated leg. In the present study, we evaluated the effect of using continuous noninvasive lower limb oximetry with near-infrared reflectance spectroscopy (NIRS) to detect tissue hypoxia and guide distal perfusion catheter (DPC) placement on the rates of leg ischemia requiring surgical intervention. METHODS: We performed a retrospective analysis of patients who had undergone femoral VA-ECLS at our institution from 2010 to 2014 (pre-NIRS era) and 2017 to 2021 (NIRS era). Patients who had undergone cannulation during the 2015 to 2016 transition era were excluded. The baseline characteristics, short-term outcomes, and ischemic complications requiring surgical intervention (eg, fasciotomy, thrombectomy, amputation, exploration) were compared across the two cohorts. RESULTS: Of the 490 patients included in the present study, 141 (28.8%) and 349 (71.2%) had undergone cannulation before and after the routine use of NIRS to direct DPC placement, respectively. The patients in the NIRS cohort had had a greater incidence of hyperlipidemia (53.7% vs 41.1%; P = .015) and hypertension (71.4% vs 60%; P = .020) at baseline, although they were less likely to have been supported with an intra-aortic balloon pump before ECLS cannulation (26.9% vs 37.6%; P = .026). These patients were also more likely to have experienced cardiac arrest (22.9% vs 7.8%; P ≤ .001) and a pulmonary cause (5.2% vs 0.7%; P = .04) as an indication for ECLS, with ECLS initiated less often for acute myocardial infarction (15.8% vs 34%; P ≤ .001). The patients in the NIRS cohort had had a smaller arterial cannula size (P ≤ .001) and a longer duration of ECLS support (5 vs 3.25 days; P ≤ .001) but significantly lower rates of surgical intervention for limb ischemia (2.6% vs 8.5%; P = .007) despite comparable rates of DPC placement (49.1% vs 44.7%; P = .427), with only two patients (1.1%) not identified by NIRS ultimately requiring surgical intervention. CONCLUSIONS: The use of a smaller arterial cannula (≤15F) and continuous NIRS monitoring to guide selective insertion of DPCs could be a valid and effective strategy associated with a reduced incidence of ischemic events requiring surgical intervention.

Trends in Unplanned Admissions of Patients With Adult Congenital Heart Disease Based on the Japanese Registry of All Cardiac and Vascular Diseases-Diagnosis Procedure Combination Study.

BACKGROUND: The prevalence of adult congenital heart disease (ACHD) is increasing rapidly and in particular, patients who underwent complicated surgeries are reaching their youth and middle age. Therefore, the need for ACHD treatment will increase, but the current medical situation is unknown. In this study we assessed trends in unplanned admissions in patients with ACHD in Japan.Methods and Results: From the Japanese Registry of All Cardiac and Vascular Diseases-Diagnosis Procedure Combination, a nationwide claim-based database, we selected patients aged >15 years with CHD defined by the International Classification of Diseases, 10th Revision codes. We identified 39,676 admissions between April 2012 and March 2018; 10,444 (26.3%) were unplanned. Main diagnoses were categorized into 3 degrees of complexity (severe, moderate, and mild) and other. Among unplanned admissions, the proportion of the severe group increased with time. Patients in the mild group were significantly older than those in the moderate and severe groups (median age: 70.0, 39.0, and 32.0 years, respectively). There were 765 deaths during hospitalization (overall mortality rate, 7.3%). The odds ratio of death during admission was significantly higher in patients aged >50 years, especially in the moderate group. CONCLUSIONS: Patients with moderate or severe ACHD tended to experience unplanned admissions at a younger age. In anticipation of greater numbers of new, severe patients, we need to prepare for their increasing medical demands.

Nationwide epidemiology and clinical practice patterns of pediatric urinary tract infections: application of multivariate time-series clustering.

BACKGROUND: The nationwide epidemiology and clinical practice patterns for younger children hospitalized with urinary tract infections (UTIs) were unclear. METHODS: We conducted a retrospective observational study consisting of 32,653 children aged < 36 months who were hospitalized with UTIs from 856 medical facilities during fiscal years 2011-2018 using a nationally representative inpatient database in Japan. We investigated the epidemiology of UTIs and changes in clinical practice patterns (e.g., antibiotic use) over 8 years. A machine learning algorithm of multivariate time-series clustering with dynamic time warping was used to classify the hospitals based on antibiotic use for UTIs. RESULTS: We observed marked male predominance among children aged < 6 months, slight female predominance among children aged > 12 months, and summer seasonality among children hospitalized with UTIs. Most physicians selected intravenous second- or third-generation cephalosporins as the empiric therapy for treating UTIs, which was switched to oral antibiotics during hospitalizations for 80% of inpatients. Whereas total antibiotic use was constant over the 8 years, broad-spectrum antibiotic use decreased gradually from 5.4 in 2011 to 2.5 days of therapy per 100 patient-days in 2018. The time-series clustering distinctively classified 5 clusters of hospitals based on antibiotic use patterns and identified hospital clusters that preferred to use broad-spectrum antibiotics (e.g., antipseudomonal penicillin and carbapenem). CONCLUSIONS: Our study provided novel insight into the epidemiology and practice patterns for pediatric UTIs. Time-series clustering can be useful to identify the hospitals with aberrant practice patterns to further promote antimicrobial stewardship. A higher resolution version of the Graphical abstract is available as Supplementary information.

Validation Study of Diabetes Definitions Using Japanese Diagnosis Procedure Combination Data Among Hospitalized Patients.

BACKGROUND: Validation studies of diabetes definitions using nationwide healthcare databases are scarce. We evaluated the validity of diabetes definitions using disease codes and antidiabetic drug prescriptions in the Japanese Diagnosis Procedure Combination (DPC) data via medical chart review. METHODS: We randomly selected 500 records among 15,334 patients who participated in the Japan Public Health Center-Based Prospective Study for the Next Generation in Yokote City and who had visited a general hospital in Akita between October 2011 and August 2018. Of the 500 patients, 98 were linked to DPC data; however, only 72 had sufficient information in the medical chart. Gold standard confirmation was performed by board-certified diabetologists. DPC-based diabetes definitions were based on the International Classification of Diseases, 10th Revision codes and antidiabetic prescriptions. Sensitivity, specificity, and the positive and negative predictive values (PPV and NPV, respectively) of DPC-based diabetes definitions were evaluated. RESULTS: Of 72 patients, 23 were diagnosed with diabetes using chart review; 19 had a diabetes code, and 13 had both a diabetes code and antidiabetic prescriptions. The sensitivity, specificity, PPV, and NPV were 89.5% (95% confidence interval [CI], 66.9-98.7%), 96.2% (95% CI, 87.0-99.5%), 89.5% (95% CI, 66.9-98.7%), and 96.2% (95% CI, 87.0-99.5%), respectively, for (i) diabetes codes alone; 89.5% (95% CI, 66.9-98.7%), 94.3% (95% CI, 84.3-98.8%), 85.0% (95% CI, 62.1-96.8%), and 96.2% (95% CI, 86.8-99.5%) for (ii) diabetes codes and/or prescriptions; 68.4% (95% CI, 43.4-87.4%), 100% (95% CI, 93.3-100%), 100% (95% CI, 75.3-100%), and 89.8% (95% CI, 79.2-96.2%) for (iii) both diabetes codes and prescriptions. CONCLUSION: Our results suggest that DPC data can accurately identify diabetes among inpatients using (i) diabetes codes alone or (ii) diabetes codes and/or prescriptions.

The Effects of Hospital Volume on Short-Term Outcomes of Laparoscopic Surgery for Rectal Cancer: A Large-Scale Analysis of 37,821 Cases on a Nationwide Administrative Database.

INTRODUCTION: Laparoscopic low anterior resection (L-LAR) has become widely accepted for the treatment of rectal cancer. However, little is known about the superiority of L-LAR in a real-world setting (including low-volume hospitals) and the association between the short-term outcomes and hospital volume focusing on L-LAR. METHODS: This is a retrospective cohort study. A total of 37,821 patients who underwent LAR for rectal cancer were analyzed using the Diagnosis Procedure Combination (DPC) database from January 2014 to December 2017. The short-term surgical outcomes were analyzed using a multilevel analysis. Hospital volumes were divided into quartiles, including low (1-31), middle (32-55), high (56-91), and very-high volume (92-444 resections per 4 years). The effects of hospital volume on the outcomes were investigated. RESULTS: The study population included 8,335 patients (22%) who underwent open low anterior resection (O-LAR) and 29,486 patients (78%) who underwent L-LAR. The in-hospital mortality and morbidity were consistent with previous reports. In patients who underwent L-LAR, the in-hospital mortality (0.12% vs. 0.41%; OR: 0.33; p = 0.005), the rate of reoperation (3.76% vs. 6.48%; OR: 0.67; p < 0.001), and the perioperative transfusion rate (3.81% vs. 5.90%; OR: 0.66; p < 0.001) were significantly lower in very-high-volume hospitals than in low-volume hospitals. These effects of hospital volume were not observed in O-LAR. CONCLUSIONS: Our present study demonstrates that high volume improves outcomes in patients who underwent L-LAR in a real-world setting.

Deciphering Structure-Function Relationship Unveils Salt-Resistant Mode of Action of a Potent MRSA-Inhibiting Antimicrobial Peptide, RR14.

Multidrug-resistant (MDR) bacteria lead to considerable morbidity and mortality, threatening public health worldwide. In particular, infections of methicillin-resistant Staphylococcus aureus (MRSA) in hospital and community settings are becoming a serious health problem. Antimicrobial peptides (AMPs) are considered novel therapeutic targets against MDR bacteria. However, salt sensitivity reduces the bactericidal potency of AMPs, posing a major obstacle for their development as antibiotics. Thus, the design and development of salt-insensitive peptides with potent antibacterial activity is imperative. Here, we employed biochemical and biophysical examinations coupled with molecular modeling to systematically investigate the structure-function relationship of a novel salt-insensitive AMP, RR14. The secondary structure of RR14 was characterized as an apparent α-helix, a structure that confers strong membrane-permeabilizing ability targeting bacterial-mimetic membranes. Additionally, the bioactive structure of RR14 was determined in complex with dodecylphosphocholine (DPC) micelles, where it possesses a central α-helical segment comprising residues R4 to K13 (R4-K13). RR14 was observed to orient itself into the DPC micelle with its N terminus and the α-helical segment (I5-R10) buried inside the micelles, which is essential for membrane permeabilization and bactericidal activity. Moreover, the specific and featured arrangement of positively charged residues of RR14 on its amphipathic helical conformation has great potential to render its strong salt resistance ability. Our study explored the structure-function relationship of RR14, explaining its possible mode of action against MRSA and other microbes. The insights obtained are of great applicability for the development of new antibacterial agents. IMPORTANCE Many antimicrobial peptides have been observed to become inactive in the presence of high salt concentrations. To further develop new and novel AMPs with potent bactericidal activity and salt insensitivity, understanding the structural basis for salt resistance is important. Here, we employed biochemical and biophysical examinations to systematically investigate the structure-function relationship of a novel salt-insensitive AMP, RR14. RR14 was observed to orient itself into DPC micelles with the N terminus and the α-helical segment (I5-R10) buried inside the micelles, which is essential for membrane permeabilization and bactericidal activity. Moreover, the specific and featured arrangement of cationic residues of RR14 on its amphipathic helical conformation renders its strong salt resistance ability. The insights obtained are of great applicability for developing new antibacterial agents.

USP11 mediates repair of DNA-protein cross-links by deubiquitinating SPRTN metalloprotease.

DNA-protein cross-links (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription, and recombination. USP11 deubiquitinase participates in DNA repair, but the role of USP11 in DPC repair is not known. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins cross-linked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN auto-proteolysis and chromatin accessibility during DPC repair. Previously, VCPIP1 and USP7 deubiquitinases have been shown to regulate SPRTN. Here, we identify USP11 as an SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination and promotes SPRTN auto-proteolysis in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings show that USP11 regulates SPRTN auto-proteolysis and SPRTN-mediated DPC repair to maintain genome stability.

The effects of mepiquat chloride (DPC) on the soluble protein content and the activities of protective enzymes in cotton in response to aphid feeding and on the activities of detoxifying enzymes in aphids.

BACKGROUND: Mepiquat chloride (DPC) enhances the resistance of cotton plants, and it is widely used as a growth regulator. DPC can stimulate photosynthesis, stabilize the structure of cotton leaves, and affect population reproduction and energy substances in Aphis gossypii Glover (cotton aphids), but interactions between DPC and cotton aphids remain unclear. In this study, we analyzed the physiological responses of cotton to DPC, and the toxicity of DPC toward cotton aphids, before and after feeding, to explore the DPC-induced defense mechanism against cotton aphids. RESULTS: Measurements of protective enzyme activity in cotton showed that the soluble protein contents, peroxidase (POD) activity, and catalase (CAT) activity in cotton treated with different concentrations of DPC were higher than in the control. Superoxide dismutase (SOD) activity was higher than that of the control when the concentration of DPC was < 0.1 g/L. Under aphid feeding stress, POD activity in cotton treated with a low insect population density was significantly lower than in the controls, but the reverse was true for cotton treated with a high insect population density, and SOD activity was positively correlated with population density. The activities of detoxification enzymes in field and laboratory experiments showed that DPC promoted the specific activity of glutathione S-transferase (GST) in cotton aphids, while the specific activities of carboxylesterase (CarE) and acetylcholinesterase (AchE) were decreased. CONCLUSIONS: DPC enhanced the aphid resistance in cotton by increasing the soluble protein content and the activity of protective enzymes. It also had a toxic effect on cotton aphids by increasing GST activity (the main DPC target). DPC increased the soluble protein content and protective enzymes activity in cotton under aphid stress, and thereby enhanced tolerance to cotton aphids. It conclude that DPC interferes with cotton aphids through indirect (DPC induced cotton defense responses) and direct (DPC toxicity to cotton aphids) ways, which plays a positive role in interfering with cotton aphids.

NMR Structure and Localization of the Host Defense Peptide ThanatinM21F in Zwitterionic Dodecylphosphocholine Micelle: Implications in Antimicrobial and Hemolytic Activity.

Non-hemolytic antimicrobial peptides (AMPs) are vital lead molecules for the designing and development of peptide-based antibiotics. Thanatin a 21-amino acid long single disulfide bonded AMP is known to be highly non-hemolytic with a limited toxicity to human cells and model animals. Thanatin demonstrates a potent antibacterial activity against multidrug-resistant Gram-negative pathogens. A single mutated variant of thanatin replaced last residue Met21 to Phe or thanatin M21F has recently been found to be more active compared to the native peptide. In order to gain mechanistic insights toward bacterial cell lysis versus non-hemolysis, here, we report atomic resolution structure and mode insertion of thanatinM21F reconstituted into zwitterionic detergent micelle by use of solution NMR spectroscopy. The 3D structure of thanatinM21F in DPC micelle is defined by an anti-parallel ß-sheet between residues I9-F21 with a central cationic loop, residues N12-R14. PRE NMR studies revealed hydrophobic core residues of thanatinM21F are deeply inserted in the DPC micelle, while residues at the extended N-terminal half of the peptide are appeared to be mostly surface localized. Marked structural differences of thanatin and thanatinM21F in negatively charged LPS and DPC micelle could be correlated with non-hemolytic and antibacterial activity.