Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST). RESULTS: Using CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity. CONCLUSIONS: Our results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.

Tracking Inhibition of Human Small C-Terminal Domain Phosphatase 1 Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.

[The experience on the construction of the cluster prevention and control system for COVID-19 infection in designated hospitals during the period of "Category B infectious disease treated as Category A"].

The COVID-19 epidemic has spread to the whole world for three years and has had a serious impact on human life, health and economic activities. China's epidemic prevention and control has gone through the following stages: emergency unconventional stage, emergency normalization stage, and the transitional stage from the emergency normalization to the "Category B infectious disease treated as Category B" normalization, and achieved a major and decisive victory. The designated hospitals for prevention and control of COVID-19 epidemic in Tianjin has successfully completed its tasks in all stages of epidemic prevention and control, and has accumulated valuable experience. This article summarizes the experience of constructing a hospital infection prevention and control system during the "Category B infectious disease treated as Category A" period in designated hospital. The experience is summarized as the "Cluster" hospital infection prevention and control system, namely "three rings" outside, middle and inside, "three districts" of green, orange and red, "three things" before, during and after the event, "two-day pre-purification" and "two-director system", and "one zone" management. In emergency situations, we adopt a simplified version of the cluster hospital infection prevention and control system. In emergency situations, a simplified version of the "Cluster" hospital infection prevention and control system can be adopted. This system has the following characteristics: firstly, the system emphasizes the characteristics of "cluster" and the overall management of key measures to avoid any shortcomings. The second, it emphasizes the transformation of infection control concepts to maximize the safety of medical services through infection control. The third, it emphasizes the optimization of the process. The prevention and control measures should be comprehensive and focused, while also preventing excessive use. The measures emphasize the use of the least resources to achieve the best infection control effect. The fourth, it emphasizes the quality control work of infection control, pays attention to the importance of the process, and advocates the concept of "system slimming, process fattening". Fifthly, it emphasizes that the future development depends on artificial intelligence, in order to improve the quality and efficiency of prevention and control to the greatest extent. Sixth, hospitals need to strengthen continuous training and retraining. We utilize diverse training methods, including artificial intelligence, to ensure that infection control policies and procedures are simple. We have established an evaluation and feedback mechanism to ensure that medical personnel are in an emergency state at all times.

Extraction of Tooth Cusps based on DBSCAN Density Clustering and Neighborhood Search Algorithm.

A good tooth cusp extraction is helpful in evaluating the effect of cosmetic dental work in virtual tooth surgery. We propose a new tooth cusp extraction, which integrates the DBSCAN (density-based spatial clustering of applications with noise) clustering algorithm with the neighborhood search algorithm to extract tooth cusp from a three-dimensional cloud-point tooth model. This method used the point cloud height and curvature to screen out the dented point set. Then we employ the DBSCAN clustering algorithm to segment different feature regions of the tooth surface and generate the candidate point set. Finally, the candidate point set was accurately located at the tooth apex through the neighborhood search algorithm and the traversal search method of non-maximum suppression. The experimental results show that the proposed method is superior to the traditional watershed algorithm-based methods by calculating the recall rate and accuracy rate, and also has higher extraction speed and extraction precision than manual extraction methods.

[Study of hospitalization risk indicators for intensive care unit patients evaluated by intelligent calculation method].

OBJECTIVE: To explore the characteristics of the changes in risk score for intensive care unit (ICU) patients during hospitalization by the intelligent calculation method, and to provide evidence for the risk prevention. METHODS: In this retrospective study, ICU patients of the Fifth Central Hospital in Tianjin from November 3, 2021 to March 28, 2022 were enrolled and divided into ≥ 14 days group, 10-13 days group, 7-9 days group, and 3-6 days group according to the ICU length of stay. Risk scores assessed by the intelligent calculation method of the ICU patients were collected, including nutritional risk screening 2002 (NRS 2002), Caprini score and Padua score. NRS 2002 score for all patients, Caprini score for surgical patients and Padua score for internal medicine patients were selected. Trends in change of each score were compared between patients admitted to ICU 1, 3, 7 (if necessary), 10 (if necessary), and 14 days (if necessary). RESULTS: A total of 138 patients were involved, including 79 males and 59 females, with an average age of (61.71±18.86) years and an average hospital stay of [6.00 (4.00, 9.25)] days. (1) in the group with ICU length of stay ≥ 14 days (21 cases): there was no significant change in the NRS 2002 scores of the patients within 10 days, but the NRS 2002 score was significantly decreased in 14 days as compared with 1 day [3.00 (2.50, 3.50) vs. 4.00 (3.00, 5.00), P < 0.05]; both Caprini and Padua score were increased with prolonged hospital stay and compared with 1 day, the scores at the other time points were significantly increased, especially at 14 days [Caprini score: 5.00 (3.25,7.00) vs. 2.50 (1.25, 5.50), Padua score: 6.00 (6.00, 7.00) vs. 3.00 (1.00, 3.00), both P < 0.05]. (2) in the group with ICU length of stay from 10-13 days (15 cases): with the prolonged hospital stay, there was no significant change in NRS 2002 score, but both Caprini and Padua score were increased at 3, 7, 10 days, especially at 10 days [Caprini score: 3.00 (2.00, 4.75) vs. 2.00 (0.25, 2.75), Padua score: 5.00 (3.50, 6.00) vs. 2.00 (0.50, 4.00), both P < 0.05]. (3) in the group with ICU length of stay from 7-9 days (23 cases): compared with 1 day, the NRS 2002 score at 3 days and7 days were decreased, but the Caprini and Padua score were increased, especially at 7 days [NRS 2002 score: 2.00 (1.00, 4.00) vs. 2.00 (2.00, 4.00), Caprini score: 3.00 (2.00, 5.50) vs. 2.00 (0.25, 3.00), Padua score: 5.00 (4.00, 6.00) vs. 2.00 (0,2.00),all P < 0.05]. (4) in the group with ICU length of stay from 3-6 days (79 cases): compared with 1 day, the NRS 2002 score at 3 days was decreased [NRS 2002 score: 2.00 (1.00, 3.00) vs. 2.00 (1.00, 3.00), P < 0.05], Caprini and Padua score were significantly increased [Caprini score: 3.00 (2.00, 4.00) vs. 2.00 (1.00, 3.00), Padua score: 5.00 (4.00, 5.00) vs. 2.00 (1.00, 3.00), both P < 0.05]. CONCLUSIONS: Based on dynamic assessment of intelligent calculation methods, the risk of thrombosis in ICU patients increased with hospital length of stay, and the nutritional risk was generally flat or reducing in different hospitalization periods.

Kinetic, Inhibition, and Structural Characterization of a Malonate Semialdehyde Decarboxylase-like Protein from Calothrix sp. PCC 6303: A Gateway to the non-Pro1 Tautomerase Superfamily Members.

The amino-terminal proline (Pro1) has long been thought to be a mechanistic imperative for tautomerase superfamily (TSF) enzymes, functioning as a general base or acid in all characterized reactions. However, a global examination of more than 11,000 nonredundant sequences of the TSF uncovered 346 sequences that lack Pro1. The majority (∼85%) are found in the malonate semialdehyde decarboxylase (MSAD) subgroup where most of the 294 sequences form a separate cluster. Four sequences within this cluster retain Pro1. Because these four sequences might provide clues to assist in the identification and characterization of activities of nearby sequences without Pro1, they were examined by kinetic, inhibition, and crystallographic studies. The most promising of the four (from Calothrix sp. PCC 6303 designated 437) exhibited decarboxylase and tautomerase activities and was covalently modified at Pro1 by 3-bromopropiolate. A crystal structure was obtained for the apo enzyme (2.35 Šresolution). The formation of a 3-oxopropanoate adduct with Pro1 provides clues to build a molecular model for the bound ligand. The modeled ligand extends into a region that allows interactions with three residues (Lys37, Arg56, Glu98), suggesting that these residues can play roles in the observed decarboxylation and tautomerization activities. Moreover, these same residues are conserved in 16 nearby, non-Pro1 sequences in a sequence similarity network. Thus far, these residues have not been implicated in the mechanisms of any other TSF members. The collected observations provide starting points for the characterization of the non-Pro1 sequences.

A small-molecule cocktail promotes mammalian cardiomyocyte proliferation and heart regeneration.

Zebrafish and mammalian neonates possess robust cardiac regeneration via the induction of endogenous cardiomyocyte (CM) proliferation, but adult mammalian hearts have very limited regenerative potential. Developing small molecules for inducing adult mammalian heart regeneration has had limited success. We report a chemical cocktail of five small molecules (5SM) that promote adult CM proliferation and heart regeneration. A high-content chemical screen, along with an algorithm-aided prediction of small-molecule interactions, identified 5SM that efficiently induced CM cell cycle re-entry and cytokinesis. Intraperitoneal delivery of 5SM reversed the loss of heart function, induced CM proliferation, and decreased cardiac fibrosis after rat myocardial infarction. Mechanistically, 5SM potentially targets α1 adrenergic receptor, JAK1, DYRKs, PTEN, and MCT1 and is connected to lactate-LacRS2 signaling, leading to CM metabolic switching toward glycolysis/biosynthesis and CM de-differentiation before entering the cell-cycle. Our work sheds lights on the understanding CM regenerative mechanisms and opens therapeutic avenues for repairing the heart.

Targeted Covalent Inhibition of Small CTD Phosphatase 1 to Promote the Degradation of the REST Transcription Factor in Human Cells.

The repressor element-1 silencing transcription factor (REST) represses neuronal gene expression, whose dysregulation is implicated in brain tumors and neurological diseases. A high level of REST protein drives the tumor growth in some glioblastoma cells. While transcription factors like REST are challenging targets for small-molecule inhibitors, the inactivation of a regulatory protein, small CTD phosphatase 1 (SCP1), promotes REST degradation and reduces transcriptional activity. This study rationally designed a series of α,ß-unsaturated sulfones to serve as potent and selective covalent inhibitors against SCP1. The compounds inactivate SCP1 via covalent modification of Cys181 located at the active site entrance. Cellular studies showed that the inhibitors inactivate SCP1 in a time- and dose-dependent manner with an EC50 ∼1.5 µM, reducing REST protein levels and activating specific REST-suppressed genes. These compounds represent a promising line of small-molecule inhibitors as a novel lead for glioblastoma whose growth is driven by REST transcription activity.

Simplicity is the Ultimate Sophistication-Crosstalk of Post-translational Modifications on the RNA Polymerase II.

The highly conserved C-terminal domain (CTD) of the largest subunit of RNA polymerase II comprises a consensus heptad (Y1S2P3T4S5P6S7) repeated multiple times. Despite the simplicity of its sequence, the essential CTD domain orchestrates eukaryotic transcription and co-transcriptional processes, including transcription initiation, elongation, and termination, and mRNA processing. These distinct facets of the transcription cycle rely on specific post-translational modifications (PTM) of the CTD, in which five out of the seven residues in the heptad repeat are subject to phosphorylation. A hypothesis termed the "CTD code" has been proposed in which these PTMs and their combinations generate a sophisticated landscape for spatiotemporal recruitment of transcription regulators to Pol II. In this review, we summarize the recent experimental evidence understanding the biological role of the CTD, implicating a context-dependent theme that significantly enhances the ability of accurate transcription by RNA polymerase II. Furthermore, feedback communication between the CTD and histone modifications coordinates chromatin states with RNA polymerase II-mediated transcription, ensuring the effective and accurate conversion of information into cellular responses.

[Effect of different fluid resuscitation strategies on renal function in patients with septic shock induced acute kidney injury].

OBJECTIVE: To compare the therapeutic effect of fluid resuscitation strategy guided by pulse-indicated continuous cardiac output (PiCCO) monitoring and early goal-directed therapy (EGDT) on renal function of acute kidney injury (AKI) patients caused by septic shock. METHODS: Septic shock patients with AKI admitted to the intensive care unit (ICU) of Tianjin Fifth Central Hospital and Teda International Cardiovascular Hospital from March 2017 to February 2020 were enrolled. All patients were given fluid resuscitation. Patients were divided into PiCCO-guided fluid resuscitation group [PiCCO group, intrathoracic blood volume index (ITBVI) was maintained between 850-1 000 mL/m2] and EGDT-guided fluid resuscitation group [EGDT group, central venous pressure (CVP) was maintained between 8-12 mmHg (1 mmHg = 0.133 kPa) or CVP ≤ 15 mmHg when patients received mechanical ventilation (MV)] according to both the patient's condition and the informed consent of the patient's family. The changes of heart rate (HR), mean arterial pressure (MAP), CVP, blood lactic acid (Lac), fluid balance, urine volume and serum creatinine (SCr) at 6, 24, and 48 hours after fluid resuscitation in the two groups were observed, and the renal replacement therapy (RRT), duration of MV, length of ICU stay and 28-day mortality between the two group were compared. RESULTS: (1) A total of 94 patients were enrolled, including 51 in the EGDT group and 43 in the PiCCO group. There was no significant difference in gender, age, acute physiology and chronic health evaluation II (APACHE II) score, sequential organ failure assessment (SOFA) score, procalcitonin (PCT), HR, MAP, CVP, Lac or SCr at ICU admission between the two groups. (2) The parameters of hemodynamics, fluid balance, urine volume and SCr were improved with the time of resuscitation in the two groups, and there was no significant difference in HR, MAP or Lac between the two groups. Compared with the EGDT group, the CVP decreased significantly at 24 hours and 48 hours after fluid resuscitation in the PiCCO group (mmHg: 9.1±0.9 vs. 12.0±1.3 at 24 hours, 8.0±1.0 vs. 10.2±1.3 at 48 hours), the fluid balance significantly decreased (mL: 2 929.8±936.3 vs. 3 898.4±923.5 at 24 hours, 3 143.5±1 325.4 vs. 4 843.8±1 326.7 at 48 hours), and the condition of urine volume and SCr were better in the PiCCO group [urine volume (mL×kg-1×h-1): 1.02±0.21 vs. 0.79±0.14 at 24 hours, 1.28±0.18 vs. 0.94±0.22 at 48 hours; SCr (µmol/L): 145.7±37.6 vs. 164.3±46.4 at 24 hours, 128.4±33.6 vs. 143.5±37.7 at 48 hours), with significant differences (all P < 0.05). (3) Compared with the EGDT group, the rate of RRT in the PiCCO group was lower [11.6% (5/43) vs. 17.6% (9/51)], the duration of MV and the length of ICU stay were shorter [duration of MV (days): 4.64±1.31 vs. 6.50±2.19, length of ICU stay (days): 10.35±3.50 vs. 14.50±5.78), with significant differences (all P < 0.05). There was no significant difference in the 28-day mortality between the PiCCO group and EGDT group [14.0% (6/43) vs. 15.7% (8/51), P > 0.05]. CONCLUSIONS: Fluid resuscitation strategy guided by PiCCO in septic shock patients with AKI can reduce the amount of fluid load, improve renal function, shorten the MV duration and length of ICU stay, and shows clinical significance.

[Retrospective analysis of the on-site treatment of the coronavirus disease 2019 epidemic on the Costa Crociere cruise].

Coronavirus disease 2019 (COVID-19) epidemic is the most widespread global pandemic in the past 100 years. Person-to-person transmission of COVID-19 infection leads to the major threat of human safety and health. At 00:00 on January 24th, 2020, Tianjin City launched the first-level response to the COVID-19 epidemic. At 18:00 on the same day, Management Committee of Dongjiang Free Trade Port Zone of Tianjin received areport that there were 15 people who had fever on the Costa Crociere carrying 4 806 people from Japan back to the home port of Tianjin Dongjiang Cruise. At the same time, there are more than 140 Chinese Hubei tourists. Tianjin Municipal Committee and Government, Tianjin Customs, Binhai New Area District Committee Government, Tianjin Health Commission, Tianjin Binhai New Area Health Commission formed an emergency command center immediately to deal with the epidemic comprehensively. At 06:40 on January 25th, 2020, the medical investigation team made up by Tianjin Binhai New Area Health Commission and Tianjin East Administration of Customs boarded the cruise ship. With reference to the customs inspection and quarantine regulations, in accordance with the Diagnosis and treatment of pneumonia caused by novel coronavirus (trial version 3) for mulated by the National Health Commission of the People's Republic of China and the Novel coronavirus infected pneumonia port control and technology plan (first version) formulated by the General Administration of Customs, combined with the actual situation of cruise ships, the medical investigation team developed the inspection standards, including door-to-door inspections, temperature measurement and epidemiological investigations on all persons on board of the cruise ship. A total of 4 806 person-times were investigated in the affected area, including 3 706 tourists and 1 100 crew members. Seventeen people at high risk of COVID-19 were identified, including three Wuhan tourists. The reports of 2019 novel coronavirus (2019-nCoV) nucleic acid detection on throat swab samples for those who were identified as high risk were returned as all negative at 14:54 on the same day. At 19:30, the medical investigation team completed the investigation and evacuated the cruise ship. The temperature measurement, medical observation and resettlement of passenger were handed over to relevant personnel. After 2 weeks, the follow-up result of 2019-nCoV nucleic acid of 17 high risk people were all negative. The overall command and comprehensive coordination of the onshore command center together with the rigid principles and excellent responds ability of the on-site epidemic investigation team ensured the successful completion of the epidemic investigation work, and also provided reference for further improving the management and disposal capacity of public health emergencies at sea.

Bacterial magnetic particles-polyethylenimine vectors deliver target genes into multiple cell types with a high efficiency and low toxicity.

Bacterial magnetic particles (BMPs) are biosynthesized magnetic nano-scale materials with excellent dispersibility and biomembrane enclosure properties. In this study, we demonstrate that BMPs augment the ability of polyethylenimine (PEI) to deliver target DNA into difficult-to-transfect primary porcine liver cells, with transfection efficiency reaching over 30%. Compared with standard lipofection and polyfection, BMP-PEI gene vectors significantly enhanced the transfection efficiencies for the primary porcine liver cells and C2C12 mouse myoblast cell lines. To better understand the mechanism of magnetofection using BMP-PEI/DNA vectors, transmission electron microscopy (TEM) images of transfected Cos-7, HeLa, and HEP-G2 cells were observed. We found that the BMP-PEI/DNA complexes were trafficked into the cytoplasm and nucleus by way of vesicular transport and endocytosis. Our study builds support for the versatile BMP-PEI vector transfection system, which might be exploited to transfect a wide range of cell types or even to reach specific targets in the treatment of disease. KEY POINTS: • We constructed a BMP-PEI gene delivery vector by combining BMPs and PEI. • The vector significantly enhanced transfection efficiencies in eukaryotic cell lines. • The transfection mechanism of this vector was explained in our study.

[A clinical study on the determination of cuff pressure in artificial airway by minimum air leakage method].

OBJECTIVE: To compare the cuff pressure and leakage volume and the related complications of filling the tracheal tube cuff by minimum air leakage method and cuff pressure manometer method after endotracheal intubation, so as to provide theoretical basis for patients who was intubated to obtain appropriate cuff pressure. METHODS: A prospective randomized controlled study was conducted. 100 patients admitted to the department of critical care medicine of the Fifth Center Hospital in Tianjin from December 2015 to June 2019 were enrolled. According to the random number table method, the patients were divided into the experimental group and control group, with 50 patients in each group. After successful endotracheal intubation, all patients were placed in a supine position with the head of the bed raised by 30 degree angle. The experimental group used the minimum air leakage method, and used the cuff pressure manometer to obtain the cuff pressure. In the control group, cuff pressure was maintained at 25-30 cmH2O (1 cmH2O = 0.098 kPa). Parameters such as cuff pressure and ventilator leakage volume at the beginning and 4 hours, 8 hours after the inflation were compared between the two groups, as well as the incidence of ventilation-associated pneumonia (VAP) and airway complications after extubation. RESULTS: Among the 100 cases, 53 were males and 47 were females. The age ranged from 23 to 87 years old, with an average of (68.53±8.46) years old. The intubation time ranged from 1 to 16 days. (1) At 4 hours and 8 hours after inflation, the cuff pressures of the two groups were lower than that of the first time of inflation, and the air leakage of the ventilator increased gradually with the extension of time. Compared with the control group, cuff pressures at each time point in the experimental group were significantly higher than those in the control group [mmHg (1 mmHg = 0.133 kPa): 33.72±9.14 vs. 25.68±5.26 at 0 hour, 30.54±7.81 vs. 24.35±4.93 at 4 hours, 26.57±5.64 vs. 22.42±4.14 at 8 hours, all P < 0.05], and ventilator leakage volumes were smaller than those in the control group (mL: 25.57±8.51 vs. 34.65±9.47 at 0 hour, 40.54±8.51 vs. 60.34±7.85 at 4 hours, both P < 0.05). (2) The incidence of VAP in the experimental group was significantly lower than that in the control group (4% vs. 10%, P < 0.05). There was no statistically significant difference in the incidence of other airway complications between the experimental group and control group (airway mucosal edema: 14% vs. 12%, ulcer: 8% vs. 6%, tracheal esophageal fistula: 0% vs. 0%, hoarseness: 4% vs. 6%, cough: 30% vs. 34%, sore throat: 28% vs. 32%, tracheal softening: 0% vs. 0%, cuff rupture: 10% vs. 8%, all P > 0.05). CONCLUSIONS: The optimal cuff pressure is very important for preventing VAP and reducing airway complications. The minimum air leakage method makes the clinical obtained endotracheal intubation cuff pressure more accurately, with less air leakage, safe and effective, and it is worthy of clinical promotion.

[Comparison of pulmonary circulation hemodynamics and respiratory mechanics induced by drowning with equal volume of freshwater and seawater in sheep: a randomized controlled study].

OBJECTIVE: To compare the effects of freshwater and seawater drowning on sheep's pulmonary circulation hemodynamics and respiratory mechanics. METHODS: According to the random number table method, healthy crossbred sheep were divided into freshwater drowning group (n = 12) and seawater drowning group (n = 12). 30 mL/kg of freshwater or seawater was infused respectively through trachea for approximately 5 minutes. Before the drowning, immediately after drowning, and 30, 60, 120 minutes after drowning, the systemic circulation hemodynamic parameters [heart rate (HR), mean arterial pressure (MAP), cardiac output (CO)] were monitored by pulse indicator continuous cardiac output (PiCCO); the respiratory parameters were obtained through the ventilator, including tidal volume (VT), lung compliance (Cdyn), oxygenation index (PaO2/FiO2), peak airway pressure (Ppeak)]; PiCCO and the right heart floating catheter (Swan-Ganz catheter) was used to measure pulmonary hemodynamic parameters [pulmonary systolic pressure (PAS), pulmonary diastolic pressure (PAD), pulmonary artery wedge pressure (PAWP), and extravascular lung water (EVLW)]. The animals were sacrificed at the end of the experiment, and the amount of residual water in the respiratory tract was measured; the pathological changes in the lung tissue were observed by hematoxylin-eosin (HE) staining. RESULTS: (1) Systemic circulation hemodynamics: compared with the values before drowning, HR, MAP, and CO at the time of immediately after drowning in both freshwater and seawater were significantly increased and peaked. In addition, all indicators in the freshwater drowning group were significantly higher than those in the seawater drowning group [HR (bpm): 170.75±1.87 vs. 168.67±2.27, MAP (mmHg, 1 mmHg = 0.133 kPa): 172.92±1.62 vs. 159.42±3.18, CO (L/min): 13.27±0.71 vs. 10.33±0.73, all P < 0.05]. (2) Respiratory parameters: compared with values before drowning, PaO2/FiO2, VT, and Cdyn decreased immediately in both freshwater and seawater drowning groups, Ppeak was significantly increased; in addition, the values in the seawater drowning group were decreased or increased more significantly than freshwater drowning group [PaO2/FiO2 (mmHg): 37.83±1.99 vs. 60.42±5.23, VT (mL): 86.25±7.66 vs. 278.75±9.67, Cdyn (mL/cmH2O): 8.86±0.33 vs. 23.02±0.69, Ppeak (cmH2O, 1 cmH2O = 0.098 kPa): 42.17±2.69 vs. 17.67±1.15, all P < 0.01]. In addition, PaO2/FiO2 in the freshwater drowning group was gradually increased over time, while the seawater group continued to decline. (3) Pulmonary circulation hemodynamic parameters: PAS, PAD, PAWP at the time of immediately after drowning in both freshwater and seawater groups were significantly higher than before drowning; in addition, the freshwater drowning group was significantly higher than the seawater drowning group [PAS (mmHg): 34.58±2.87 vs. 26.75±1.66, PAD (mmHg): 27.25±1.22 vs. 16.75±0.87, PAWP (mmHg): 27.83±1.85 vs. 11.75±1.82, all P < 0.01]. Thereafter, PAS and PAD in the freshwater drowning group gradually decreased, while the parameters in the seawater drown group continued to increase. PAWP gradually decreased after freshwater or seawater drowning, and recovered to pre-drowning levels 120 minutes after drowning and 30 minutes after drowning, respectively. EVLW continued to increase after freshwater drowning, reaching a peak at 30 minutes, and then decreased, until 120 minutes after drowning was still significantly higher than that before drowning (mL/kg: 10.73±1.27 vs. 7.67±0.69, P < 0.01); EVLW could not be measured. (4) Residual water in the respiratory tract: residual water in the freshwater drowning group was significantly less than that in the seawater drowning group (mL: 164.33±25.21 vs. 557.33±45.23, P < 0.01). (5) HE staining: partial alveolar atrophied in the freshwater drowning group, some alveolar spaces were broken, alveolar spaces and alveolar cavity showed a little powdery substance deposition; it was noted that alveolar expanded in the seawater drowning group, alveolar spaces were broken and bleeding and edema were obvious in the interstitial space. CONCLUSIONS: The effect of seawater drowning on the respiratory mechanics and pulmonary circulation of animals is more obvious than that of freshwater drowned animals, and the amount of residual water in the respiratory tract is also significantly more than that of freshwater drowned animals.

Cell-Wide Survey of Amide-Bonded Lysine Modifications by Using Deacetylase CobB.

BACKGROUND: Lysine post-translational modifications are important regulators of protein function. Proteomic and biochemical approaches have resulted in identification of several lysine modifications, including acetylation, crotonylation, and succinylation. Here, we developed an approach for surveying amide-bonded lysine modifications in the proteome of human tissues/cells based on the observation that many lysine modifications are amide-bonded and that the Salmonella enterica deacetylase, CobB, is an amidase. RESULTS: After the proteome of human tissues/cells was denatured and the non-covalently bonded metabolites were removed by acetone washes, and the amide-bonded modifiers were released by CobB and analyzed using liquid- and/or gas chromatography/mass spectrometry metabolomic analysis. This protocol, which required 3-4 days for completion, was used to qualitatively identify more than 40 documented and unreported lysine modifications from the human proteome and to quantitatively analyze dynamic changes in targeted amide-bonded lysine modifications. CONCLUSIONS: We developed a method that was capable of monitoring and quantifying amide-bonded lysine modifications in cells of different origins.

Mapping RNAPII CTD Phosphorylation Reveals That the Identity and Modification of Seventh Heptad Residues Direct Tyr1 Phosphorylation.

The C-terminal domain (CTD) of the largest subunit in eukaryotic RNA polymerase II has a repetitive heptad sequence of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 which is responsible for recruiting transcriptional regulatory factors. The seventh heptad residues in mammals are less conserved and subject to various post-translational modifications, but the consequences of such variations are not well understood. In this study, we use ultraviolet photodissociation mass spectrometry, kinetic assays, and structural analyses to dissect how different residues or modifications at the seventh heptad position alter Tyr1 phosphorylation. We found that negatively charged residues in this position promote phosphorylation of adjacent Tyr1 sites, whereas positively charged residues discriminate against it. Modifications that alter the charges on seventh heptad residues such as arginine citrullination negate such distinctions. Such specificity can be explained by conserved, positively charged pockets near the active sites of ABL1 and its homologues. Our results reveal a novel mechanism for variations or modifications in the seventh heptad position directing subsequent phosphorylation of other CTD sites, which can contribute to the formation of various modification combinations that likely impact transcriptional regulation.

Tyr1 phosphorylation promotes phosphorylation of Ser2 on the C-terminal domain of eukaryotic RNA polymerase II by P-TEFb.

The Positive Transcription Elongation Factor b (P-TEFb) phosphorylates Ser2 residues of the C-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II and is essential for the transition from transcription initiation to elongation in vivo. Surprisingly, P-TEFb exhibits Ser5 phosphorylation activity in vitro. The mechanism garnering Ser2 specificity to P-TEFb remains elusive and hinders understanding of the transition from transcription initiation to elongation. Through in vitro reconstruction of CTD phosphorylation, mass spectrometry analysis, and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we uncover a mechanism by which Tyr1 phosphorylation directs the kinase activity of P-TEFb and alters its specificity from Ser5 to Ser2. The loss of Tyr1 phosphorylation causes an accumulation of RNA polymerase II in the promoter region as detected by ChIP-seq. We demonstrate the ability of Tyr1 phosphorylation to generate a heterogeneous CTD modification landscape that expands the CTD's coding potential. These findings provide direct experimental evidence for a combinatorial CTD phosphorylation code wherein previously installed modifications direct the identity and abundance of subsequent coding events by influencing the behavior of downstream enzymes.

[Effect of alveolar macrophages phagocytosis on prognosis in patients with acute respiratory distress syndrome caused by abdominal infection: a multicenter study].

OBJECTIVE: To investigate the effects of alveolar macrophage phagocytosis on prognosis in patients with acute respiratory distress syndrome (ARDS) caused by abdominal infection. METHODS: ARDS patients caused by severe intra-abdominal infection admitted to intensive care unit (ICU) of Tianjin Fourth Central Hospital, Tianjin Nankai Hospital, Tianjin First Central Hospital and Tianjin Fifth Central Hospital from June 2016 to March 2018 were enrolled. The gender, age, acute physiology and chronic health evaluation II (APACHE II) within 24 hours of admission, neutral red phagocytosis and alkaline phosphatase activity of macrophages in bronchoalveolar lavage fluid, the length of ICU stay, total hospitalization time, hospitalization expenses, and prognosis were recorded. According to the prognosis, the patients were divided into death group and survival group, and the parameters were compared between the two groups. Pearson test was used to analyze the correlation between neutral red phagocytosis function of macrophages and alkaline phosphatase activity and other indicators. The prognosis was analyzed by binary Logistic regression combined with neutral red phagocytosis and alkaline phosphatase activity in patients, and the predictive value of both subjects on prognosis was analyzed by the receiver operating characteristic (ROC) curve. RESULTS: Twenty patients were enrolled in the study, with 8 in the death group and 12 in the survival group. Compared with the survival group, the death group was older (years old: 58.50±14.86 vs. 46.67±13.40), APACHE II score was higher (21.50±3.93 vs. 13.58±4.12), neutral red phagocytosis ability and alkaline phosphatase activity of alveolar macrophages were significantly decreased (A value: 0.265±0.050 vs. 0.338±0.016; µmol/L: 12.06±1.24 vs. 17.96±3.90), and the length of ICU stay was significantly longer (days: 22.00±14.59 vs. 11.50±3.17), hospitalization cost was significantly increased (10 thousand Yuan: 24.17±11.02 vs. 13.44±3.53), the total hospitalization time was shorter (days: 25.25±15.01 vs. 35.67±8.58), and the difference was statistically significant (all P < 0.05). There was no significant difference in gender between the survival group and the death group [male (case): 8 vs. 6, P > 0.05]. The neutral red phagocytosis ability of alveolar macrophages in ARDS patients caused by abdominal infection was negatively correlated with age, APACHE II score and the length of ICU stay (r value was -0.328, -0.572, -0.809, respectively, all P < 0.05); alkaline phosphatase activity was negatively correlated with age, APACHE II score, the length of ICU stay and hospitalization expenses (r value was -0.334, -0.583, -0.470, -0.517, respectively, all P < 0.05). Binary Logistic regression analysis showed that neutral red phagocytosis [odds ratio (OR) = 0.596, 95% confidence interval (95%CI) = 0.212-0.997] and alkaline phosphatase activity (OR = 0.573, 95%CI = 0.339-0.968) were the influencing factors of prognosis (both P < 0.05). ROC curve analysis showed that the AUC of neutral red phagocytosis ability for prognosis of ARDS patients caused by abdominal infection was 0.948, and the sensitivity and specificity were 91.7% and 87.5% when the off-cut value was 0.317. The AUC of alkaline phosphatase for the prognosis of ARDS patients caused by abdominal infection was 0.813; when the cut-off value was 19.72 µmol/L, the sensitivity was 75.0%, and the specificity was 87.5%. CONCLUSIONS: The alveolar macrophage phagocytosis dysfunction in ARDS patients caused by severe abdominal infection was not only related to the severity of the disease, but also increased the medical burden of patients, and significantly affected the mortality of such patients.

Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription.

The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser5 (pSer5) is gradually dephosphorylated by CTD phosphatases, whereas Ser2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer2 and pSer5 CTDs. We found that Ssu72 dephosphorylates pSer5 effectively but only has low activities toward pSer7 and pSer2 The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight ß-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr1 next to Ser2, prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr1 further prohibited Ssu72 binding to pSer2 and thereby prevented untimely Ser2 dephosphorylation. Our results reveal critical roles for Tyr1 in differentiating the phosphorylation states of Ser2/Ser5 of CTD in RNA polymerase II that occur at different stages of transcription.

BLT1 in dendritic cells promotes Th1/Th17 differentiation and its deficiency ameliorates TNBS-induced colitis.

Leukotriene B4 (LTB4) synthesis is enhanced in the colonic mucosa in patients with inflammatory bowel disease (IBD). BLT1, a high-affinity receptor for LTB4, exhibits no effect on the progression of dextran sodium sulfate (DSS)-induced colitis, which mostly relies on innate immunity. Here, we reported that BLT1 regulates trinitrobenzene sulfonic acid (TNBS)-induced colitis, which reflects CD4+ T-cell-dependent adaptive immune mechanisms of IBD. We found that BLT1 signaling enhanced the progression of colitis through controlling the production of proinflammatory cytokines by dendritic cells (DCs) and modulating the differentiation of Th1 and Th17. BLT1-/- mice displayed an alleviated severity of TNBS-induced colitis with reduced body weight loss and infiltrating cells in the lamina propria. BLT1 deficiency in DCs led to reduced production of proinflammatory cytokines, including IL-6, TNF-α, and IL-12, and these results were further confirmed via treatment with a BLT1 antagonist. The impaired cytokine production by BLT1-/- DCs subsequently led to reduced Th1 and Th17 differentiation both in vitro and in vivo. We further performed a conditional DC reconstitution experiment to assess whether BLT1 in DCs plays a major role in regulating the pathogenesis of TNBS-induced colitis, and the results indicate that BLT1 deficiency in DCs also significantly reduces disease severity. The mechanistic study demonstrated that BLT1-regulated proinflammatory cytokine production through the Gαi ßγ subunit-phospholipase Cß (PLCß)-PKC pathway. Notably, we found that treatment with the BLT1 antagonist also reduced the production of proinflammatory cytokines by human peripheral blood DCs. Our findings reveal the critical role of BLT1 in regulating adaptive immunity and TNBS-induced colitis, which further supports BLT1 as a potential drug target for adaptive immunity-mediated IBD.