Preoperative evaluation of gastric cancer and risk factors for postoperative complications / 中华胃肠外科杂志

Surgical resection plays pivotal role in the treatment of gastric cancer. Adequate preoperative evaluation, precise intraoperative maneuver and delicate postoperative management lay the foundation for successful gastrectomy. The aim of preoperative evaluation is to stage tumor and identify potential risk factors (including preoperative factors like age, ASA status, body mass index, comorbidity, hypoalbuminemia, and intraoperative factors like blood loss and combined resection) which could lead to postoperative complication. With the management of prehabilitation, adequate medical decision could be made and patient's fast recovery could be ensured. With the rapid adoption of ERAS concept, there is increasing attention to prehabilitation which focus on optimization of cardio-pulmonary capacity and muscular-skeletal capacity. Despite of the efficacy of prehabilitation demonstrated by randomized controlled trials, consensus has yet to be reached on the following items: specific intervention, optimal measurement, candidate population and optimal timing for intervention. Balancing the efficiency and safety, preoperative evaluation could be put into clinical practice smoothly.

Effect of Echinococcus multilocularis infection on Tim3 expression in spleen natural killer cells of mice / 中国血吸虫病防治杂志

OBJECTIVE@#To investigate the effect of Echinococcus multilocularis infection on Tim3 expression and its co-expression with immune checkpoint molecules 2B4 and LAG3 in spleen natural killer (NK) cells of mice.@*METHODS@#C57BL/6 mice, each weighing (20 ± 2) g, were randomly divided into a high-dose infection group (15 mice), a low-dose infection group (13 mice), and a control group (11 mice). Mice in the high- and low-dose infection groups were inoculated with 2 000 and 50 Echinococcus multilocularis protoscolices via the hepatic portal vein, while animals in the control group was injected with an equivalent amount of physiological saline via the hepatic portal vein. Mouse spleen cells were harvested 12 and 24 weeks post-infection, and Tim3 expression and its co-expression with 2B4 and LAG3 in NK cells were detected using flow cytometry.@*RESULTS@#There were significant differences in the proportions of Tim3 expression (F = 13.559, P < 0.001) and Tim3 and 2B4 co-expression (F = 12.465, P < 0.001) in mouse spleen NK cells among groups 12 weeks post-infection with E. multilocularis, and the proportion of Tim3 expression was significantly higher in mouse spleen NK cells in the low-dose infection group [(23.84 ± 2.28)%] than in the high-dose infection group [(15.72 ± 3.67)%] and the control group [(16.14 ± 3.83)%] (both P values < 0.01), while the proportion of Tim3 and 2B4 co-expression was significantly higher in mouse spleen NK cells in the low-dose infection group [(22.20 ± 2.13)%] than in the high-dose infection group [(14.17 ± 3.81)%] and the control group [(15.20 ± 3.77)%] (both P values < 0.01). There were significant differences in the proportions of Tim3 expression (F = 5.243, P < 0.05) and Tim3 and 2B4 co-expression (F = 4.659, P < 0.05) in mouse spleen NK cells among groups 24 weeks post-infection with E. multilocularis infection, and the proportions of Tim3 expression and Tim3 and 2B4 co-expression were significantly lower in mouse spleen NK cells in the high-dose infection group [(20.55 ± 7.04)% and (20.98 ± 7.12)%] than in the control group [(31.38 ± 3.19)% and (31.25 ± 3.06)%] (both P values < 0.05), and there were no significantly difference between the proportions of Tim3 expression and Tim3 and 2B4 co-expression in splenic NK cells in the low-dose infection group [(26.80 ± 6.47)% and (26.48 ± 6.48)%] and the control group (both P > 0.05). There were no significant differences in the proportions of Tim3 and LAG3 co-expression in mouse spleen NK cells among groups 12 (F = 2.283, P > 0.05) and 24 weeks post-infection (F = 0.375, P > 0.05). In the low-dose infection group, there were no significant differences in the proportions of Tim3 expression or Tim3 and 2B4 co-expression in mouse spleen NK cells 12 (t = -1.137, P > 0.05) or 24 weeks post-infection (t = -1.658, P > 0.05), and the proportion of Tim3 and LAG3 co-expression increased in mouse spleen NK cells 24 weeks post-infection relative to 12 weeks post-infection (t = -5.261, P < 0.01). In the highdose infection group, there was no significant difference in the proportion of Tim3 expression in mouse spleen NK cells 12 and 24 weeks post-infection (t = -1.546, P > 0.05); however, the proportions of Tim3 co-expression with 2B4 and LAG3 increased in mouse splenic NK cells 24 weeks post-infection relative to 12 weeks post-infection (t = -2.425 and -4.745, both P values < 0.05).@*CONCLUSIONS@#The Tim3 expression and Tim3 co-expression with LAG3 and 2B4 on spleen NK cells is affected by doses of E. multilocularis infection and disease stages, and present different phenotypes during the course of alveolar echinococcosis. NK cells tend to form an immunosuppressive phenotype with the progression of E. multilocularis infection, which facilitates immune escape and chronic parasitism of E. multilocularis.

Clinicopathological features of olfactory carcinoma / 中华病理学杂志

Objective: To investigate the clinicopathological features and differential diagnosis of olfactory carcinoma (OC). Methods: Twenty-one cases of sinonasal tumors, including those initially diagnosed as olfactory neuroblastoma (ONB) and those with uncertain diagnosis, were collected from the Department of Pathology, the First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) from January 2016 to August 2022, among which 3 cases were reclassified as OC. The clinicopathological features were investigated, and the remaining 18 cases were used as control. Results: Of the three OC patients, 2 were male and 1 was female, with an average age of 57 years ranging from 35 to 74 years. Microscopically, the tumor cells were arranged in solid, nested or lobulated patterns with occasional palisading around the solid nests. The stroma was highly vascular with focal neurofibrillary areas. There were prominent rosettes or pseudorosettes formation. The tumor cells were mainly ovoid to spindly with scant to moderate amount of cytoplasm, one or several small nucleoli, and fine chromatin content. Brisk mitotic figures were seen. In all 3 cases of OC, there were scanty atypical glands and some were ciliated. Immunohistochemically, at least one epithelial marker and neuroendocrine marker were diffusely expressed in the tumor. Some of the tumor cells were positive for p40 and p63, and the sustentacular cells showed the expression of S-100 protein. All cases tested were negative for NUT, CD99 and desmin, with intact expression of SMARCA4 (BRG1) and SMARCB1 (INI-1). Ki-67 proliferation index varied from 20% to 80%. Follow-up after 16-18 months showed no mortality with tumor recurrence from 1 patient after 16 months. Conclusion: OC is a rare sinonasal tumor with neuroepithelial differentiation, its histomorphology is diverse, and the combination of immunohistochemical markers is essential for appropriate diagnosis.

Factors influencing super-long hospital stays in patients undergoing radical gastrectomy in the age of enhanced recovery after surgery / 中华胃肠外科杂志

Objective: To obtain experience and generate suggestions for reducing average hospital stays, optimizing perioperative management of patients with gastric cancer and improving utilization of medical resources by analyzing the factors influencing super-long hospital stays in patients undergoing radical gastrectomy in the age of enhanced recovery after surgery (ERAS). Methods: This was a case-control study. Inclusion criteria: (1) pathologically diagnosed gastric adenocarcinoma; (2) radical surgery for gastric cancer; and (3) complete clinicopathologic data. Exclusion criteria: (1) history of upper abdominal surgery; (2) presence of distant metastasis of gastric cancer or other ongoing neoplastic diseases; (3) concurrent chemoradiotherapy; and (4) preoperative gastric cancer-related complications such as obstruction or perforation. The study cohort comprised 285 eligible patients with hospital stays of ≥30 days (super-long hospital stay group). Using propensity score matching in a 1:1 ratio, age, sex, medical insurance, pTNM stage, and extent of surgical resection as matching factors, 285 patients with hospital stays of < 30 days during the same period were selected as the control group (non-long hospital stay group). The primary endpoint was relationship between pre-, intra-, and post-operative characteristics and super-long hospital stays. Clavien-Dindo grade was used to classify complications. Results: Univariate analysis showed that number of comorbidities, number of preoperative consultations, preoperative consultation, inter-departmental transference, operation time, open surgery, blood loss, intensive care unit time, presence of surgical or non-surgical complications, Clavien-Dindo grade of postoperative complications, and reoperation were associated with super-long hospital stays (all P<0.05). Inter-departmental transference (OR=4.876, 95% CI: 1.500-16.731, P<0.001), preoperative consultation time ≥ 3 d (OR=1.758, 95%CI: 1.036-2.733, P=0.034), postoperative surgery-related complications (OR = 6.618, 95%CI: 2.141-20.459, P=0.01), and higher grade of complications (Clavien-Dindo Grade I: OR = 7.176, 95%CI: 1.785-28.884, P<0.001; Clavien-Dindo Grade II: OR = 18.984, 95%CI: 6.286-57.312, P<0.001; Clavien-Dindo Grade III-IV: OR=7.546, 95%CI:1.495-37.952, P=0.014) were independent risk factors for super-long hospital stays. Conclusion: Optimizing preoperative management, enhancing perioperative management, and surgical quality control can reduce the risk of prolonging average hospital stay.

Effects of High-frequency Repetitive Transcranial Magnetic Stimulation to Premotor Areas on Upper Limb Motor Dysfunction after Stroke / 中国康复理论与实践

Objective:To explore the effect of high-frequency repetitive transcranial magnetic stimulation (rTMS) on upper limb motor function in hemiplegic patients after stroke. Methods:From August, 2018 to July, 2019, 60 patients with hemiplegia after stroke were randomly divided into control group (n = 30) and observation group (n = 30). Both groups received conventional treatment. The observation group accepted 5 Hz rTMS to ipsilesional hemisphere premotor areas for three weeks. The control group received sham stimulation. They were assessed with Fugl-Meyer Assessment-Upper Extremities (FMA-UE), Brunnstrom stages, modified Ashworth Scale (MAS), modified Barthel Index (MBI) and Wolf Motor Function Test before and after treatment. Results:Two patients dropped in the control group. After treatment, the scores of FMA-UE, MBI and Wolf Motor Function Test improved in both groups (|t| > 3.686, P < 0.01), and the difference values of FMA-UE and Wolf Motor Function Test before and after treatment were more in the observation group than in the control group (|t| > 2.119, P < 0.05). Conclusion:High-frequency rTMS to ipsilesional hemisphere premotor areas could improve the recovery of upper limb and hand motor function in hemiplegic patients after stroke.

Cases diagnosis of imported malaria in Jiangsu province, 2014-2016 / 中华流行病学杂志

Objective: To understand the situation related to health seeking and diagnosis of imported malaria and to provide practical measures for malaria elimination in Jiangsu province. Methods: Data on imported malaria cases in Jiangsu province was retrieved in CISDCP from 2014 to 2016. Relevant information on health seeking behavior, diagnosis and treatment of the disease was gathered. Results: A total of 1 068 imported cases were reported in Jiangsu province from 2014 to 2016. Except for one malaria case that was caused by blood transfusion, the rest patients were all recognized as 'imported'. Majority of the cases were migrant laborers working in African countries. The accurate rates on the diagnosis of ovale, vivax and quartan malaria and mixed infection were relatively low, as 79.3% (107/135), 29.5% (18/61), 52.9% (18/34) and 0.0% (0/2) at the primary health care settings, respectively. Rate of seeking health care on the same day of onset was more in 2015 than in 2014 and 2016 (χ(2)=18.6, P=0.001). While only 65.4% (699/1 068) of the patients were diagnosed correctly at the primary health care settings. There appeared no statistical difference in the 3-year-study period (χ(2)=5.4, P=0.246). Capacity on 'correct diagnosis' seemed stronger at the CDC than at the hospital levels (χ(2)=13.2, P=0.000; χ(2)=5.4, P=0.020). Totally, 72.7% (32/44) of the severe falciparum malaria cases did not immediately seek for health care when the symptoms started. Conclusions: Migrant workers returning from the high endemic malaria areas seemed to have poor awareness in seeking health care services. Capability on correct diagnosis for malaria at the primary health care settings remained unsatisfactory and staff from these settings needs to receive adequate training.

Associations of obesity and physical activity with cognition in people aged 50 and above in Shanghai / 中华流行病学杂志

Objective: To investigate the associations of obesity and physical activity with cognition in the elderly. Methods: A cross-sectional survey was conducted from October 2009 to June 2010 among people aged ≥50 years selected through multistage random cluster sampling in Shanghai. The subjects' body weight, body height, waist circumference and hip circumference were measured to calculate body mass index (BMI) and waist-hip ratio (WHR), and the data on self-reported physical activity level were collected through questionnaire survey. A comprehensive battery of cognitive tests was conducted to assess subjects' cognitive functions, including verbal recall, forward digit span (FDS), backward digit span (BDS), and verbal fluency (VF). General linear model was used to examine the associations of BMI, WHR and physical activity with cognition. Results: A total of 7 913 participants were included, with a median age of 60 years. Age, sex, education level, income level, BMI, WHR and physical activity level were significantly associated with cognitive scores in univariate analysis. After adjusted for age, sex, education level and income level, BMI was no longer significantly associated with cognitive scores in all cognitive functions (all P>0.01). WHR was significantly associated with VF score (P<0.01). Abdominally obese participants had lower VF score than non-abdominally obese participants (P<0.01). Physical activity level was significantly associated with all cognitive functions (P<0.01). Compared with participants with moderate physical activity level, participants with low physical activity level had lower scores in all cognitive functions (P<0.01). Conclusion: Abdominal obesity and low physical activity level were negatively associated with cognition level in the elderly, suggesting that waist circumference control and physical activity might help maintain cognition in the elderly.

Interactive effects between health literacy and mobile phone dependence as well as its relation with unintentional injuries in middle school students / 中华流行病学杂志

Objective: To investigate the interaction between health literacy, mobile phone dependence and unintentional injuries in middle school students, and to provide guidance for prevention on unintentional injuries in adolescents. Methods: From November 2015 to January 2016, a questionnaire survey was conducted among 22 628 middle school students in Shenyang of Liaoning province, Bengbu of Anhui province, Xinxiang of Henan province, Ulanqab of Inner Mongolia Autonomous Region, Chongqing and Yangjiang of Guangdong province. Chinese Adolescent Interactive Health Literacy Questionnaire (CAIHLQ), Self-rating Questionnaire for Adolescent Problematic Mobile Phone Use (SQAPMPU), and Unintentional Injuries Assessment Scale and demographic variables were used to measure the health literacy, mobile phone dependence and unintentional injuries among the Chinese middle school students. Results: The detection rates of mobile phone dependence and unintentional injuries were 25.4% and 46.7%, respectively. The rates of unintentional injuries of middle school students with mobile phone dependence and with low, medium and high health literacy were 53.6%, 44.4% and 48.8%, 48.1%, 41.7%. Factors as mobile phone dependence, low and middle health literacy were positively related to unintentional injuries (OR=1.452, 1.196, 1.364). However, the multiplicative interaction between mobile phone dependence and health literacy on unintentional injuries was noticed significant (OR=1.217, 95%CI: 1.041-1.422). Conclusions: Our results showed that the prevalence of unintentional injuries was relatively high in middle school students. Health literacy and mobile phone dependence seemed related to unintentional injuries. Interaction between health literacy and mobile phone dependence on unintentional injuries appeared significant.

Thermodynamics of ABC transporters

ABC transporters form the largest of all transporter families, and their structural study has made tremendous progress over recent years. However, despite such advances, the precise mechanisms that determine the energy-coupling between ATP hydrolysis and the conformational changes following substrate binding remain to be elucidated. Here, we present our thermodynamic analysis for both ABC importers and exporters, and introduce the two new concepts of differential-binding energy and elastic conformational energy into the discussion. We hope that the structural analysis of ABC transporters will henceforth take thermodynamic aspects of transport mechanisms into account as well.

How does transmembrane electrochemical potential drive the rotation of Fo motor in an ATP synthase?

While the field of ATP synthase research has a long history filled with landmark discoveries, recent structural works provide us with important insights into the mechanisms that links the proton movement with the rotation of the Fo motor. Here, we propose a mechanism of unidirectional rotation of the Fo complex, which is in agreement with these new structural insights as well as our more general ΔΨ-driving hypothesis of membrane proteins: A proton path in the rotor-stator interface is formed dynamically in concert with the rotation of the Fo rotor. The trajectory of the proton viewed in the reference system of the rotor (R-path) must lag behind that of the stator (S-path). The proton moves from a higher energy site to a lower site following both trajectories simultaneously. The two trajectories meet each other at the transient proton-binding site, resulting in a relative rotation between the rotor and stator. The kinetic energy of protons gained from ΔΨ is transferred to the c-ring as the protons are captured sequentially by the binding sites along the proton path, thus driving the unidirectional rotation of the c-ring. Our ΔΨ-driving hypothesis on Fo motor is an attempt to unveil the robust mechanism of energy conversion in the highly conserved, ubiquitously expressed rotary ATP synthases.

Proton transfer-mediated GPCR activation

G-protein coupled receptors (GPCRs) play essential roles in signal transduction from the environment into the cell. While many structural features have been elucidated in great detail, a common functional mechanism on how the ligand-binding signal is converted into a conformational change on the cytoplasmic face resulting in subsequent activation of downstream effectors remain to be established. Based on available structural and functional data of the activation process in class-A GPCRs, we propose here that a change in protonation status, together with proton transfer via conserved structural elements located in the transmembrane region, are the key elements essential for signal transduction across the membrane.

Prevalence of clinical hip abnormalities in haemophilia A and B: an analysis of the UDC database.

Clinical hip abnormalities, secondary to recurrent joint and/or muscle bleeding in persons with haemophilia, have not been well characterized and have the potential for significant morbidity. We aimed to examine the prevalence of clinical hip abnormalities in the US haemophilia population and to explore associations between these findings and putative risk factors. We conducted a study of hip abnormalities of 8192 subjects aged 2-69 years with haemophilia A and haemophilia B (54% of haemophilia A and haemophilia B are severe) currently enrolled in the Universal Data Collection (UDC) database. Associations between hip abnormality and type/severity of haemophilia A/B, current age, history of high-titre (≥ 5 BU) inhibitor (HTinh), concomitant ankle (AA) and knee arthropathy (KA), overweight and obesity and prophylaxis were examined using logistic regression. Overall prevalence of hip abnormality at the last recorded UDC visit for all subjects was 16.7%. Haemophilia A (aOR = 1.3, 1.0-1.4), severe haemophilia (aOR = 1.3, 1.0-1.5), a history of HTinh (aOR = 1.4, 1.1-1.7), and concomitant AA (aOR = 1.7, 1.4-1.9) were each independently associated with hip abnormality. Older age (45-69 years) was significantly associated with hip abnormality prevalence only in subjects with KA (aOR = 3.4, 1.9-5.9). The presence of overweight (aOR = 1.4, 1.1-1.8) and obesity (aOR = 2.1, 1.6-2.8) was associated with hip abnormality only among subjects without KA. Hip abnormality prevalence was not influenced by prophylaxis (aOR = 0.9, 0.8-1.1). These data suggest that hip abnormalities in US patients with haemophilia are associated with haemophilia severity and type, HTinh, concomitant AA and, depending on the presence or absence of KA, advancing age and obesity.

GPCR activation: protonation and membrane potential

GPCR proteins represent the largest family of signaling membrane proteins in eukaryotic cells. Their importance to basic cell biology, human diseases, and pharmaceutical interventions is well established. Many crystal structures of GPCR proteins have been reported in both active and inactive conformations. These data indicate that agonist binding alone is not sufficient to trigger the conformational change of GPCRs necessary for binding of downstream G-proteins, yet other essential factors remain elusive. Based on analysis of available GPCR crystal structures, we identified a potential conformational switch around the conserved Asp2.50, which consistently shows distinct conformations between inactive and active states. Combining the structural information with the current literature, we propose an energy-coupling mechanism, in which the interaction between a charge change of the GPCR protein and the membrane potential of the living cell plays a key role for GPCR activation.

An open conformation determined by a structural switch for 2A protease from coxsackievirus A16

Coxsackievirus A16 belongs to the family Picornaviridae, and is a major agent of hand-foot-and-mouth disease that infects mostly children, and to date no vaccines or antiviral therapies are available. 2A protease of enterovirus is a nonstructural protein and possesses both self-cleavage activity and the ability to cleave the eukaryotic translation initiation factor 4G. Here we present the crystal structure of coxsackievirus A16 2A protease, which interestingly forms hexamers in crystal as well as in solution. This structure shows an open conformation, with its active site accessible, ready for substrate binding and cleavage activity. In conjunction with a previously reported "closed" state structure of human rhinovirus 2, we were able to develop a detailed hypothesis for the conformational conversion triggered by two "switcher" residues Glu88 and Tyr89 located within the bll2-cII loop. Substrate recognition assays revealed that amino acid residues P1', P2 and P4 are essential for substrate specificity, which was verified by our substrate binding model. In addition, we compared the in vitro cleavage efficiency of 2A proteases from coxsackievirus A16 and enterovirus 71 upon the same substrates by fluorescence resonance energy transfer (FRET), and observed higher protease activity of enterovirus 71 compared to that of coxsackievirus A16. In conclusion, our study shows an open conformation of coxsackievirus A16 2A protease and the underlying mechanisms for conformational conversion and substrate specificity. These new insights should facilitate the future rational design of efficient 2A protease inhibitors.

Structure analysis of the extracellular domain reveals disulfide bond forming-protein properties of Mycobacterium tuberculosis Rv2969c

Disulfide bond-forming (Dsb) protein is a bacterial periplasmic protein that is essential for the correct folding and disulfide bond formation of secreted or cell wallassociated proteins. DsbA introduces disulfide bonds into folding proteins, and is re-oxidized through interaction with its redox partner DsbB. Mycobacterium tuberculosis, a Gram-positive bacterium, expresses a DsbA-like protein ( Rv2969c), an extracellular protein that has its Nterminus anchored in the cell membrane. Since Rv2969c is an essential gene, crucial for disulfide bond formation, research of DsbA may provide a target of a new class of anti-bacterial drugs for treatment of M.tuberculosis infection. In the present work, the crystal structures of the extracellular region of Rv2969c (Mtb DsbA) were determined in both its reduced and oxidized states. The overall structure of Mtb DsbA can be divided into two domains: a classical thioredoxin-like domain with a typical CXXC active site, and an α-helical domain. It largely resembles its Escherichia coli homologue EcDsbA, however, it possesses a truncated binding groove; in addition, its active site is surrounded by an acidic, rather than hydrophobic surface. In our oxidoreductase activity assay, Mtb DsbA exhibited a different substrate specificity when compared to EcDsbA. Moreover, structural analysis revealed a second disulfide bond in Mtb DsbA, which is rare in the previously reported DsbA structures, and is assumed to contribute to the overall stability of Mtb DsbA. To investigate the disulphide formation pathway in M.tuberculosis, we modeled Mtb Vitamin K epoxide reductase (Mtb VKOR), a binding partner of Mtb DsbA, to Mtb DsbA.

Crystal structures and biochemical studies of human lysophosphatidic acid phosphatase type 6

Lysophosphatidic acid (LPA) is an important bioactive phospholipid involved in cell signaling through Gprotein-coupled receptors pathways. It is also involved in balancing the lipid composition inside the cell, and modulates the function of lipid rafts as an intermediate in phospholipid metabolism. Because of its involvement in these important processes, LPA degradation needs to be regulated as precisely as its production. Lysophosphatidic acid phosphatase type 6 (ACP6) is an LPA-specific acid phosphatase that hydrolyzes LPA to monoacylglycerol (MAG) and phosphate. Here, we report three crystal structures of human ACP6 in complex with malonate, L-(+)-tartrate and tris, respectively. Our analyses revealed that ACP6 possesses a highly conserved Rossmann-foldlike body domain as well as a less conserved cap domain. The vast hydrophobic substrate-binding pocket, which is located between those two domains, is suitable for accommodating LPA, and its shape is different from that of other histidine acid phosphatases, a fact that is consistent with the observed difference in substrate preferences. Our analysis of the binding of three molecules in the active site reveals the involvement of six conserved and crucial residues in binding of the LPA phosphate group and its catalysis. The structure also indicates a water-supplying channel for substrate hydrolysis. Our structural data are consistent with the fact that the enzyme is active as a monomer. In combination with additional mutagenesis and enzyme activity studies, our structural data provide important insights into substrate recognition and the mechanism for catalytic activity of ACP6.

Current trends of nonclinical safety evaluation for new drugs / 中国药理学与毒理学杂志

Nonclinical safety evaluation plays a critical role in the process of new drug development.International Conference on Harmonization (ICH) guideline M3 (R2) provides a key direction for the nonclinical safety evaluation process.Proper strategies and toxicological studies should be considered together to move the drug candidates forward efficiently and quickly to support clinical plans and market registration.Updates on ICH guidelines,such as ICH S6 and ICH S9,have great impact on the direction of development.With the increasing cost of development and competition in the industry,elements like predictivity,animal models,and regulatory compliance are also very important in the process.Therefore,an insight into all these factors is essential to toxicologists in the safety evaluation process.The ability to use the overall knowledge will result in a quicker and better new drug development program.

Structural study of the Cdc25 domain from Ral-specific guanine-nucleotide exchange factor RalGPS1a

The guanine-nucleotide exchange factor (GEF) RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP, thus regulating various downstream cellular processes. RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain, followed by a PXXP motif and a C-terminal pleckstrin homology (PH) domain. The Cdc25 domain of RalGPS1a, which shares about 30% sequence identity with other Cdc25-domain proteins, is thought to be directly engaged in binding and activating the substrate Ral protein. Here we report the crystal structure of the Cdc25 domain of RalGPS1a. The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1. The most remarkable difference between these three Cdc25 domains lies in their active sites, referred to as the helical hairpin region. Consistent with previous enzymological studies, the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding. A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex. However, analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.

Crystal structure of cytotoxin protein suilysin from Streptococcus suis

Cholesterol-dependent cytolysins (CDC) are pore forming toxins. A prototype of the CDC family members is perfringolysin O (PFO), which directly binds to the cell membrane enriched in cholesterol, causing cell lysis. However, an exception of this general observation is intermedilysin (ILY) of Streptococcus intermedius, which requires human CD59 as a receptor in addition to cholesterol for its hemolytic activity. A possible explanation of this functional difference is the conformational variation between the C-terminal domains of the two toxins, particularly in the highly conserved undecapeptide termed tryptophan rich motif. Here, we present the crystal structure of suilysin, a CDC toxin from the infectious swine pathogen Streptococcus suis. Like PFO, suilysin does not require a host receptor for hemolytic activity; yet the crystal structure of suilysin exhibits a similar conformation in the tryptophan rich motif to ILY. This observation suggests that the current view of the structure-function relationship between CDC proteins and membrane association is far from complete.

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

Nascent polypeptide associated complex (NAC) and its two isolated subunits, αNAC and βNAC, play important roles in nascent peptide targeting. We determined a 1.9 Å resolution crystal structure of the interaction core of NAC heterodimer and a 2.4 Å resolution crystal structure of αNAC NAC domain homodimer. These structures provide detailed information of NAC heterodimerization and αNAC homodimerization. We found that the NAC domains of αNAC and βNAC share very similar folding despite of their relative low identity of amino acid sequences. Furthermore, different electric charge distributions of the two subunits at the NAC interface provide an explanation to the observation that the heterodimer of NAC complex is more stable than the single subunit homodimer. In addition, we successfully built a βNAC NAC domain homodimer model based on homologous modeling, suggesting that NAC domain dimerization is a general property of the NAC family. These 3D structures allow further studies on structure-function relationship of NAC.