Exploring the drug-drug interactions might be beneficial to find active substances and develop new targeted drugs. This study aimed at exploring the change of arsenic concentration in mice and across MDCK-MDR1 cells and the cytotoxicity on K562 cells when realgar and indigo naturalis were combined. In the presence or absence of indigo naturalis, pharmacokinetics and cell-based permeability assays were used to evaluate the change of arsenic concentration, and K562 cell line was applied to evaluate the change of cytotoxicity. The drug concentration-time profiles exhibited that the combination medication group generated higher AUC, thalf, and longer MRT for arsenic, compared with the single administration of realgar. The apparent permeability coefficients (Papp) of bidirectional transport in MDCK-MDR1 cell permeability experiments showed that arsenic permeability obviously went up when indigo naturalis was incubated together. The combination medication significantly decreased the cell viability of K562 cells when both the concentration of realgar and the concentration of indigo naturalis were nontoxic. The pharmacokinetic research, the MDCK-MDR1 based permeability study, and the K562 cytotoxicity study were united together to verify the combination medication of realgar and indigo naturalis enhanced the absorption and the permeability across cells for arsenic and effectively inhibited the proliferation of K562 cell line. The molecular binding of As4S4 and indirubin was analyzed by computational study. It is predicted that the formation of the complex [As4S4…Indirubin] involves noncovalent interaction that changes the concentration of arsenic.Realgar and Throughout this paper, bond lengths are given in Å angstroms, total energies and BSSE correction in \, zero-point energies, and binding energies in kcal/mol. All calculations are performed with Gaussian 09 program package , and BSSE of complex [As4S4…Indirubin] at the HF/6-31G∗, B3LYP/6-31G∗, and wB97XD/6-31+G∗ levels of theory are shown in 4S4…Indirubin] is a local minimum with all real frequencies. As shown in ∗, B3LYP/6-31G∗, and wB97XD/6-31+G∗ levels of theory, respectively, which is larger than the sum of the covalent radii of the As and O atoms (1.84Å), but shorter than the sum of the van der Waals radii of the As and O atoms (3.37Å), indicating that the α-As4S4 and indirubin (C16H10N2O2) may form a complex of [As4S4…Indirubin] by the noncovalent interaction. The predicted binding energy with ZPE and BSSE corrections is 0.3, 0.3, and -5.8 kcal/mol at the HF/6-31G∗, B3LYP/6-31G∗, and wB97XD/6-31+G∗ levels of theory, respectively, listed in ∗ level of theory is more reasonable than the other two.Total energies with zero-point energy (ZPE) of As4S4 and indirubin form a noncovalent interaction through linking As and O atoms. It is predicted that the noncovalent interaction between As4S4 and indirubin raises the solubility and improves the releasing degree into bloodstream of As4S4 owing to the organic characteristics of indirubin.Computational study shows that As indigo naturalis enhanced the arsenic AUC in blood. This represented the fact that the arsenic of combination group had higher bioavailability in mice bodies. Realgar had higher Cmax, while it had shorter thalf and MRT, which meant realgar reached the peak concentration in a short time and it was eliminated in a relatively short period. Multiple oral administration study future illustrated that the combination medication of realgar and indigo naturalis enhanced the arsenic concentration in blood. The MDCK-MDR1 based permeability experiments verified the transport rate and penetration amount of arsenic increased when two drugs were used together in both A-B and B-A pathways. Indigo naturalis enhanced the arsenic penetration amount and permeability rate. The MDCK-MDR1 based permeability results were consistent with the pharmacokinetic study. Cytotoxicity study proved that the combination therapy efficiently decreased the survival of K562 cell line, while the cell viability was not decreased during the single medication at the same single concentration. These experiments suggested that the combination administration of realgar and indigo naturalis could produce synergistic action. An apoptotic study found that R and RI reduce the viability of K562 cells through inducing cells apoptosis and the combination medication of realgar and indigo naturalis increases the apoptotic effect of K562 cells. A theoretical result was provided by the computational work that the formation of the complex [As4S4…Indirubin] which involves noncovalent interaction might be another reason that promotes the arsenic concentration.The phenomenon which occurred in the pharmacokinetic study illustrated that the combination medication of realgar and indigo naturalis are both traditional oral drugs for treating various types of leukemia, commonly being combination medication. Starting with the combinatorial modes in traditional medicines to find mechanisms has advantages of sufficient clinical experiences accumulated in hundreds of years [Realgar andof years . United of years .


The data presented here are related to the research paper entitled “Economic Analysis of Natural Forest Disturbances: A Century of Research” . Natural disturbances have always affected forest ecosystems, altering or disrupting the flows of goods  and services  provided by forests to human societies. Economic analysis can help private or public decision-makers take forest policy decisions by understanding the causes and consequences of forest disturbances, as well as evaluating tradeoffs in alternative policy scenarios. In consequence, the economic literature about natural disturbances is very rich and diversified.This paper describes a bibliographic database gathering some 340 scientific papers related to the economic analysis of forest natural disturbances. Papers have been inventoried primarily thanks to searches on databases  using specific keywords and the dataset have been completed by searching through literature-cited sections of papers. Relevant papers have been manually encoded into a database (Excel file) taking into consideration each type of hazard  and different economic approaches. Data cover papers published in English from 1916 to 2014. Specifications TableValue of the data•Data cover 340 scientific articles in the field of economic analysis of forest disturbances.•Data allow for literature reviews in the field of economic analysis of forest disturbances either by hazard or by economic approach.•Data were used in 1The database is an original Excel file that includes some 340 scientific papers dealing with the economic analysis of forest natural disturbances. Data cover papers published in English from 1916 to 2014. Each line of the sheet represents an article; each column of the sheet represents a variable. Papers are encoded in the database according to 3 types of variables: variables concerning the references of the article; variables specifying the case study, if any; variables providing information on the method used by the author(s). 2•Wildfire: wildfire OR fire OR prescribed burning OR fuel management OR fuel treatment OR fuel reduction OR fuel break•Pest: pest OR epidemic OR infestation OR insect•Pathogen: pathogen OR disease OR infection•Storm: storm OR hurricane OR tornado OR cyclone OR typhoon OR wind OR windstorm OR windthrow OR gale OR straight line wind•Wildlife damage: browsing OR debark•Snow – Ice: snow OR ice•Other abiotic disturbances: drought OR flood OR landslide OR volcano•Other, multiple hazards: catastrophe OR damage OR mortality OR disturbance OR hazard OR risk OR stochastic OR uncertainty.We conducted a systematic literature research during the spring of 2014 using combinations of three keywords on four databases/search engines: JSTOR, ScienceDirect, Ingentaconnect and NRC Research Press. Each search combined the keywords for searching in papers’ full-text as follows: Forest* AND Economic* AND specific keyword related to the analyzed disturbance. For each hazard, we tried the following specific keywords:Complementarily, we used the reference lists of the identified papers to add other relevant articles into the database. We only include articles that are published in English. We then collected 340 papers. The comprehensive list is presented in Note that we did not find any article using the defined keywords for the category “Other abiotic disturbances”. Thus, such disturbances  do not appear in the database.“Other, multiple hazards” category gathers papers that we cannot classify into the previous ones because they deal with non-specified natural hazard or with several natural hazards or with another type of hazard . See


A virus-like particle (VLP) is an excellent tool for a compound delivery system due to its simple composition, symmetrical structure and self-assembly. Its surface modification both chemically and genetically is established, leading to the target-specific delivery and improved encapsulation efficiency. However, its physical stabilities against many harsh conditions that guarantee long term storage and oral administration have been much less studied.E. coli. Their physical properties against three strong physical conditions including long term storage (0–30 days) in 4 °C, physical stabilities against broad ranged pH (4–9) and against three types of digestive enzymes were tested. Disassembly and reassembly of VLPs for encapsidating an enhanced green fluorescent protein tagged plasmid DNA (EGFP-VLPs) were controlled by the use of reducing agent (DTT) and calcium specific chelating agent (EGTA). Lastly, delivering ability of EGFP-VLPs was performed in vivo by intramuscular injection and traced the expression of GFP in the shrimp tissues 24 hr post-injection.IHHNV-VLPs were reconstructed from recombinant IHHNV capsid protein in Upon its purification, IHHNV-VLPs were able to be kept at 4 °C up to 30 days with only slight degradation. They were very stable in basic condition (pH 8–9) and to a lesser extent in acidic condition (pH 4–6) while they could stand digestions of trypsin and chymotrypsin better than pepsin. As similar with many other non-enveloped viruses, the assembly of IHHNV-VLPs was dependent on both disulfide bridging and calcium ions which allowed us to control disassembly and reassembly of these VLPs to pack EGFP plasmid DNA. IHHNV-VLPs could deliver EGFP plasmids into shrimp muscles and gills as evident by RT-PCR and confocal microscopy demonstrating the expression of GFP in the targeted tissues.There are extensive data in which capsid proteins of the non-enveloped viruses in the form of VLPs are constructed and used as nano-containers for therapeutic compound delivery. However, the bottleneck of its application as an excellent delivery container for oral administration would rely solely on physical stability and interacting ability of VLPs to the host cells. These properties are retained for IHHNV-VLPs reported herein. Thus, IHHNV-VLPs would stand as a good applicable nanocontainer to carry therapeutic agents towards the targeting tissues against ionic and digestive conditions via oral administration in aquaculture field. Recently, many studies have focused on nano-containers designed from non-enveloped viral capsid proteins, so-called virus-like particles (VLPs). Specifically, their capability of encapsulating therapeutic cargoes, either protein-based or nucleotide-based, have rendered them potentially powerful tools for many advanced applications . MoreoveMacrobrachium rosenbergii noda virus-like particle (MrNV-VLPs) has been in a laboratory trial and paved a way for shrimp viral infection halting . After screening, the positive clones were transfected into pET-17b expression vector  for protein expression in E. coli - Rosetta strain. The crude E. coli extracts were purified by a Ni-NTA affinity chromatography , eluted by an imidazole-based elution buffer and dialyzed against Tris buffer saline  as described earlier (The IHHNV virus capsid gene was amplified from ORF3 of the IHHNV virus genome (with an estimated size of 990 bp). The specific primers for IHHNVcap (F: 5′-CATATGTGCG CCGATTCAACAAGAGCAA-3′ and R: 5′-CTCGAGTTAATGATGATGATGATGATGGTT AGTATGCATAACATAACATTTG-3′) were designed and used to amplify the gene using a gradient PCR method. The reaction conditions of PCR were one cycle at 94 °C for 5 min, followed by one set of 94 °C (30 s), 52 °C (1 min), and 72 °C (1 min) with a final extension step at 72 °C (10 min). The PCR product was ligated into pGEM-T Easy cloning vector , and transfected into  earlier . The purWe tested the physical stability of IHHNV capsid proteins against three separated conditions including (1) long-term storage at 4 °C for 1–90 days; (2) a broad range of pH (4–9); and (3) digestion by gastrointestinal enzymes . Approximately 1 mg/ml of IHHNV-VLPs were re-suspended in TBS buffer (pH 7.8) and the suspension was allowed to stand in the refrigerator (4 °C) for the periods of 1 day, 30 days, and 90 days. Degradation of the purified protein band was monitored by SDS-PAGE and silver staining. For a broad pH range tests, the protein pellets after ultracentrifugation were re-suspended in either citrate buffer (pH 4.0 and adjusted to pH 5 and 6 by 1N NaOH) or in TBS (pH 7.8 and adjusted to the desired pH by 1N HCl or 1N NaOH). The VLP samples were maintained in the given pH at 4 °C for 24 hr and the reaction was stopped by adding a loading buffer into the samples and the protein profiles were resolved by SDS-PAGE as mentioned below.For enzymatic digestion tests, IHHNV capsid proteins were divided into four groups as follows: (1) 1:50 (v/v) cocktail inhibitor (as negative control); 2 and (3) 30 mU trypsin and chymotrypsin in TBS, pH 7.8 ; and 4) 30 mU pepsin in citrate buffer, pH 4.0 (Sigma). The mixtures were allowed to stand for 1 h at room temperature and the enzymatic reaction was stopped by adding SDS-PAGE loading buffer into the mixtures. All testing samples were denatured at 95 °C and then resolved by 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) followed by a silver staining.2 in TBS) was added at 10-time volume of the disassembling buffer, followed by incubation at room temperature for 1 hr. Both disassembled and reassembled IHHNV-VLPs were washed and collected using an ultracentrifugation . The VLP pellets were then re-suspended in TBS and dropped onto a 200-mesh, carbon activated, formvar- coated grid  for 30 s. The excess suspension was blotted away by filter paper and the adhered VLPs were stained with 1% uranyl acetate for 30 s followed by visualization under a FEI Tecnai 20 transmission electron microscope operated at 80 kV.Disassembly and reassembly of VLPs was followed the protocol described previously by In order to encapsidate a large sized Pie1-DNA vector tagged with an enhanced green fluorescent protein (EGFP), we followed DNA encapsidation protocol reported earlier for IHHNV-VLPs  with somE. coli cells with or without (for cell incubation) protease inhibitor cocktail (1:50 (v/v)); (2) 400 ng of the purified IHHNV-VLPs; (3) IHHNV-VLPs storing at 4 °C for 1–90 days; (4) IHHNV-VLPs subjected to a broad range of pH (4–9) treatments; and (5) IHHNV-VLPs treated with 30 mU trypsin, chymotrypsin and pepsin. All proteins were heated for 5 min at 95 °C. The denatured proteins were resolved in a 12.5% SDS-PAGE under a reducing condition. The separated proteins were subjected to silver staining using a kit from Invitrogen .SDS-PAGE was performed according to Laemli’s method (1970). The following sets of proteins were resolved: (1) 10 µg of crude extracts from P.vanamei (n = 15 shrimp/group). One-day post injection (p.i.), the administered shrimp were anesthetized on iced water until no movement was detected. Shrimp tissues including gill, muscle and hepatopancreas were collected and analyzed for the presence of EGFP-plasmid DNA in the shrimp tissues via RT-PCR as well as detected for the fluorescent signals under confocal microscopy. In an alternative experiment, the antibody against GFP was also used to verify the presence of GFP in the tissues of administered shrimp. For RT-PCR, pair of primers used to detect GFP gene were (F) 5′-CATGGTCCTGCTGGAGTTCGTG-3′ and(R) 5′-CGTCGCCGTCCAGCTCGACCAG-3′. The PCR amplification condition was followed the PCR steps mentioned above.Animal handling protocol was followed the guidelines of the Animal Care Committee, Mahidol University (reference no. MU-IACUC 2016/013). Approximately 10 µg in 100 µl of the IHHNV-VLPs encapsulating Pie1-EGFP plasmid DNA  were injFor confocal microscopy, the tissues of the VLP administered shrimp were fixed with 4% paraformaldehyde, immersed in 30% sucrose and solidified in a Sakura TissueTek medium . After freeze-plunging in liquid nitrogen, the tissues were sectioned at 20 µm, blocked with 5% BSA, 0.1% Tween 20 in TBS and subsequently stained with 1:5,000 rabbit anti-GFP  in the blocking solution. The tissues were further exposed to 1:2,000 goat anti-rabbit IgG conjugated with Alexa593  and counterstained with 4′, 6-diamidino-2-phynylindole dihydrochloride  and visualized with an Olympus FV10i confocal laser scanning microscope. Image acquisition was performed in a Kalman line-by-line scanning mode using laser diode (LD) lines . The emission filters used was the 5-nm band pass filters with wavelengths of 460 nm (blue fluorescence), 520 nm (green fluorescence) and 630 nm (red fluorescence).Proteins from the tissues of the shrimp, both injected with EGFP-IHHNV-VLPs or unloaded IHHNV-VLPs, were extracted by a lysis buffer . Protein concentration was measured by a Bradford’s protein assay kit  and subjected to SDS-PAGE as mentioned above. The resolved proteins were transferred to nitrocellulose membrane and subjected to anti-GFP probing in the same conditions described for confocal microscopy  kit and developed on a Hyperfilm .E. coli (Rosetta) which has been shown to facilitate the expression of capsid proteins within 24 h yielding 2–3 mg/ml of the purified IHHNV capsid proteins. Through a single-step Nikel affinity chromatography, a highly purified (>95% purity) IHHNV capsid protein at the molecular mass of 37.5 kDa was obtained from the crudely prepared E. coli extracts (The IHHNV capsid protein was designed from ORF3 of IHHNV gene leading to a 990 bp construct with six histidine (His) flanking at the C-terminus . After iextracts . A minorWe further tested the physical properties of IHHNV-VLPs against three harsh conditions including long-term storage in a cold atmosphere, pH gradient, and strong hydrolytic milieu . Generally, our results demonstrated that the readily formed IHHNV-VLPs were very stable upon their storage in TBS suspension at 4 °C. The 37.5 kDa band in SDS-PAGE appeared relatively constant among all samples studied  possess a property to encapsidate the host-cell derived genomic DNA and RNA (400–600 bp) into the VLP’s cavity (E. coli (Rosetta). In the presence of 10 mM EGTA  together with DTT, the VLPs became highly dispersed, disorganized and clumped in some areas—there was no mulberry-like structure remained in all EM visualizing fields that we scanned  successfully encapsidated plasmid DNA into the VLP cavity which was revealed by the presence of 3.1 kbp band. The unloaded plasmid often showed a minor band of 6.2 kbp which assumed to be the dimerized complex of vector DNA (lane 2). It should be mentioned here that non-encapsidated VLPs showed no nucleotide band (lane 3), confirming a minimal nucleotide contamination in IHHNV-VLPs prepared from this E. coli strain as mentioned above. Our results thus implicate that inclusion of calcium ions into the suspension eases the control of VLP disassembly and reassembly processes which, in turns, facilitate the encapsidation of tagged plasmid DNA into the VLP interior.The other important physical property of VLP that renders it an applicable nano-container is the controllable VLP disassembly/reassembly. It has been reported that IHHNV-VLPs expressed s cavity . We furts cavity , we thuss cavity . However scanned . With thspension . In addiP. vannamei. Upon 24-hr administration of EGFP-VLPs into the shrimp, we could detect a positive PCR product of GFP gene (600 bp) in the gill and muscular tissues of the injected shrimp  showed minimal green fluorescence in the tissues –only the blue fluorescence of DNA staining were detected (‘Hepatopancrease’ row).We further confirmed IHHNV infectivity in the target tissues by confocal microscopy . An inteE. coli (BL21-DE3) at 16 °C for 48 hr which spontaneously form icosahedral VLPs. They have also observed that the expressed IHHNV-VLPs could encapsidate E. coli derived genomic DNA or some RNA materials (with the size of 400–600 bp) into VLP interior. In this study, we used E. coli (Rosetta) to express IHHNV capsid proteins to further extrapolate the VLP physical stability against many harsh conditions as well as to encapsidate the larger sized (3.1 kbp) plasmid DNA tagged with EGFP. Moreover, the ability of IHHNV-VLPs to deliver EGFP-plasmid in situ towards shrimp tissues is one of the highlight in our findings. We found that production of recombinant IHHNV-VLPs in E. coli (Rosetta) offered somewhat faster production rate (within 24 h in LB broth) and relatively higher protein yield in the same culture period which has been used before for MrNV capsid protein production ; however, an appreciable amount of capsid proteins (about 95%) still remained after encountering the VLPs to the moderate and strong acidic conditions (pH 4–6). In addition, the VLPs were rather stable against trypsin and chymotrypsin which are two major constitutive digestive enzymes in the alimentary tract although it is less stable against pepsin. More interestingly, the stability of IHHNV-VLPs against long-term storage or having a slow spontaneous degradation over monthly storage would offer them an outstanding nano-container that is highly practical for field trials . IHHNV-VT = 1 (in the empty VLPs) to be T = 3 (in the native virions). This surface topological difference between intact virions and VLPs casts a doubt whether the configuration of protruding domain that is responsible for viral binding to the host receptor should be affected to some certain extent. One known structural-base mechanism that is associated with the alteration of capsid subunit organization is the interaction between nucleotide sequences with the nucleic acid binding domain (Binding that leads to internalization of the virions during their infection of many non-enveloped shrimp viruses is dependent on the specific protruding domain that is usually localized on the 5-fold axis of the capsid surface where the DE loops from the five subunits meet . It has g domain  that is g domain  which isg domain . The expg domain . Regardl2 to regulate VLP reassembly. Finally, IHHNV-VLPs retained the virion surface inherent property of interacting with the shrimp tissues in vivo. Together, we believe that IHHNV-VLPs should now be another step closer to the field application as a ready-mix suspension for oral administration or submersion trials to fight against many viral infections.We have reported here the excellent physical properties of recombinant IHHNV-VLPs beyond other known non-enveloped viruses derived VLPs. They could endure many strong ionic and digestive conditions that pave a way to their application as an oral administration-based nano-container. Their encapsidation of plasmid DNA and presumably other therapeutic materials could be controlled through the use of DTT + EGTA to regulate VLP disassembly and exogenous CaCl


Presence of preoperative sarcopenia is a risk factor for postoperative complications. However, there are few reports on the presence of sarcopenia and its characteristics following gastrectomy. Sarcopenia is closely related to quality of life in elderly people. To date, the main purpose of follow-up after gastrectomy is surveillance for early detection of recurrence and secondary cancer. However, henceforth, quality of life in elderly gastric cancer patients after gastrectomy must also be evaluated. The present study aimed to investigate sarcopenia during a 1-year postoperative course in elderly gastric cancer patients and examine their characteristics. The subjects were 50 patients aged ≥70 years who underwent laparoscopy-assisted distal gastrectomy for gastric cancer and who experienced no recurrence 1 year postoperatively. Height, weight, serum albumin levels, food intake amount, grip strength, gait speed, visceral fat area, and appendicular skeletal muscle mass index were measured preoperatively and 6 months and 1 year postoperatively. Sarcopenia, obesity, and visceral obesity were diagnosed. Compared with preoperatively, indicators other than height decreased 6 months postoperatively. Compared with 6 months postoperatively, body weight, amount of food intake, and visceral fat area increased by 1 year postoperatively, unlike appendicular skeletal muscle mass index. The frequency of sarcopenia increased 6 months postoperatively compared with preoperatively; this frequency remained almost unchanged 1 year postoperatively compared with 6 months postoperatively. Further, the frequency of visceral obesity increased 1 year postoperatively compared with 6 months postoperatively. Weight increased after > 6 months postoperatively; however, most of the weight increase was in terms of fat and not muscle. We emphasize the importance of considering postoperative sarcopenia and visceral obesity. In particular, sarcopenia and visceral obesity should be carefully monitored after increases in body mass index and food consumption. In recent years, sarcopenia has been defined as the condition in which muscle mass, muscle strength, and physical ability decline , 2. SarcThe average life expectancy is increasing, and the number of older gastric cancer patients is increasing. Approximately half of the cases of patients who undergo gastric cancer surgery are early cancer, and the 5-year survival rate after early gastric cancer surgery is > 95% in Japan . TherefoThe subjects were 50 patients aged ≥70 years who underwent laparoscopy-assisted distal gastrectomy for the radical treatment of gastric cancer between November 2009 and October 2015 at National Hospital Organization Hamada Medical Center. Surgery was performed in accordance with the Japanese gastric cancer treatment guidelines 20104 (ver.4) . Lymphad2. InBody720  was used as the bioelectrical impedance analyzer to measure skeletal muscle mass. The patients were positioned in a standing posture, thereby facilitating close contact of a total of eight electrodes with both the hands and feet. Weak, noninvasive alternating current flows from these electrodes. The impedance of each limb and fuselage was measured, and the appendicular muscle mass of each limb was estimated from this impedance . The mean postoperative course at the time of the 6-month postoperative examination was 6.6 ± 0.82 months and that at time of the 1-year postoperative examination was 1.2 ± 0.95 years. The preoperative weight of the patients was significantly decreased at 6 months postoperatively and significantly increased at 1 year postoperatively. The preoperative BMI significantly decreased at 6 months postoperatively and significantly increased at 1 year postoperatively. No significant differences were noted for grip strength or serum albumin levels between 6 months and 1 year postoperatively. Gait speed significantly decreased at 6 months postoperatively .The preoperative visceral fat area was significantly decreased at 6 months postoperatively and significantly increased at 1 year postoperatively. The preoperative SMI was significantly decreased at 6 months postoperatively and showed a slightly increasing trend during the 1-year postoperative period. A comparison of the SMI at 6 months and 1 year postoperatively did not reveal any significant difference .Dietary intake is expressed as percentage values when the preoperative levels were adjusted to 100%, which significantly decreased to 78% 6 months postoperatively and significantly increased at 1 year postoperatively .With regard to weight, gait speed, BMI, SMI, and visceral fat area, there were significant differences between men and women at the same measurement time .In men, the preoperative SMI was less than the cut-off value in 9.4% of the patients; their preoperative SMI values remained less than the cut-off value at 6 months postoperatively. In 25% of the patients, the SMI was below the cut-off value at 6 months postoperatively .In women, the preoperative SMI was less than the cut-off value in 11% of the patients. Similar to that in men, the preoperative SMI in these patients was less than the cut-off value at 6 months postoperatively. The SMI dropped immediately below the cut-off value in 5.5% of the patients at 6 months postoperatively .In men, the gait speed was ≤ 0.8 m/s in 34.3% of the patients. Among these, SMI was less than the cut-off value in 72.7% of the patients at 1 year postoperatively .In women, the gait speed was ≤ 0.8 m/s in 16.6% of the patients. The SMI in these patients was below the cut-off value at 1 year postoperatively .p = 0.821) and women  rather than other elderly groups and setting the cut-off value as  . TherefoSarcopenic obesity is a state in which sarcopenia and obesity coexist and has recently been gaining attention in relation to metabolic syndrome . It is aAlthough the diagnosis of obesity is often judged based on BMI, BMI does not reveal an increase in body fat or a decrease in skeletal muscle mass , 23. Fur2 were defined as having visceral obesity; this is the cut-off value of visceral fat accumulation for diagnosing metabolic syndrome in Japan [Unlike the method of measuring muscle mass, the method for measuring visceral fat accumulation has not yet been standardized, and there are no standard values for Japanese people. Moreover, visceral fat has different mean and median values depending on race, sex, age, etc. The Guidelines for the Management of Obesity Disease 2016 recommends the use of computed tomography for the measurement of the visceral fat area at the umbilical level . In the in Japan .After distal gastrectomy, stomach volume as well as food consumption decreases, thereby resulting in weight loss during the perioperative period , which wIn the present study, compared with preoperative values, the SMI decreased 6 months postoperatively. If the muscle mass decrease until 6 months postoperatively can be inhibited, secondary sarcopenia can be prevented. In recent years, the concept of enhanced recovery after surgery has spread to patients undergoing gastrectomy, and nutritional therapy and rehabilitation from the early postoperative period are recommended . No diffDuring the perioperative period, as a result of stress associated with treatment, protein catabolism is promoted and protein assimilation is suppressed. This is a defense reaction of the body against stress, which remains difficult to control . FurtherIn the present study, the frequency of sarcopenia did not change 1 year postoperatively compared with 6 months postoperatively, but an increasing visceral obesity was noted. At 6 months postoperatively, food intake was increased and the decrease in body weight was halted. A comparison of the findings at 6 months and 1 year postoperatively revealed a significant increase in visceral fat mass, but no significant increase in SMI was noted. In other words, the increase in weight over the period from 6 months to 1 year postoperatively is mostly accounted in terms of fat. Therefore, the state of having sarcopenia and visceral obesity was found to be increasing at 1 year postoperatively. These values cannot be judged based on BMI and amount of food intake, and it is necessary to measure skeletal muscle and abdominal visceral fat.It has been reported that resistance training is effective for sarcopenia in elderly people , 36. FurTo date, the main purpose of follow-up after gastrectomy for gastric cancer has been surveillance for early detection of recurrence and secondary cancer. However, in recent years, attention has also been paid to cope with postgastrectomy symptoms and overcome nutritional issues . Few stu


Hypocretin 1 and hypocretin 2 (orexin A and B) regulate sleep, wakefulness and emotion. Tumour necrosis factor alpha (TNF‐α) is an important neuroinflammation mediator. Here, we examined the effects of TNF‐α treatment on hypocretin expression in vivo and behaviour in mice. TNF‐α decreased hypocretin 1 and hypocretin 2 expression in a dose‐dependent manner in cultured hypothalamic neurons. TNF‐α decreased mRNA stability of prepro‐hypocretin, the single precursor of hypocretin 1 and hypocretin 2. Mice challenged with TNF‐α demonstrated decreased expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in hypothalamus. In response to TNF‐α, prepro‐hypocretin mRNA decay was increased in hypothalamus. TNF‐α neutralizing antibody restored the expression of prepro‐hypocretin, hypocretin 1 and hypocretin 2 in vivo in TNF‐α challenged mice, supporting hypocretin system can be impaired by increased TNF‐α through decreasing hypocretin expression. Repeated TNF‐α challenge induced muscle activity during rapid eye movement sleep and sleep fragmentation, but decreased learning, cognition and memory in mice. TNF‐α neutralizing antibody blocked the effects of TNF‐α; in contrast, hypocretin receptor antagonist enhanced the effects of TNF‐α. The data support that TNF‐α is involved in the regulation of hypocretin expression, sleep and cognition. The findings shed some lights on the role of neuroinflammation in neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. The effects of TNF‐α on hypocretin expression in vivo, muscle activity during REM sleep (RBD), learning, cognition and memory were examined in mice. The findings support an important role of TNF‐α in the regulation of hypocretin system, sleep, and cognition and learning. The findings also shed some lights on the role of neuroinflammation on some aspects of neurodegenerative diseases including Alzheimer's disease and Parkinson's disease.22.1The following purified antibodies were purchased: hypocretin 1 , hypocretin 2 , beta‐actin (β‐actin) , prepro‐hypocretin , P38  and phospho‐p38 . Recombinant tumour necrosis factor alpha (TNF‐α) was obtained from R&D Systems. TNF‐α neutralizing antibody was purchased from BD Biosciences and Cell Signaling . Rat IgG was purchased from Jackson ImmunoResearch . The hypocretin (orexin) receptor 1 (HcrtR1/OX1R) antagonist SB334867 was purchased from Tocris (United Kingdom). All other reagents were purchased from Cell Signaling, Thermo Fisher Scientific , Sigma‐Aldrich or Bio‐Rad.2.2Protein production was determined by Western blot analysis as described by us previously.2.3® Green PCR Master Mix (Applied Biosystems) using the Bio‐Rad iQ5 Real‐Time PCR Detection System, according to the manufacturer's instructions. The quantitative RT‐PCR was performed using 4 µL of the synthesized cDNA, 12.5 µL of SYBR® Green PCR Master Mix, 1 µL of 10 µmol/L primer mix and 7.5 µL of water in a final 25 µL volume. Samples were assayed in triplicate, and the values were normalized to the relative amounts of beta‐actin mRNA level. Primer sequences were as described.Expression of prepro‐hypocretin mRNA was determined by real‐time quantitative RT‐PCR as described by us previously.2.4Prepro‐hypocretin mRNA stability and half‐life were determined by culturing primary murine hypothalamic neuron cells in medium with actinomycin D (10 μg/mL) to block transcription as described by us previously.2.5l‐glutamine, 4.5 mg/mL glucose, 100 µg/mL gentamicin and 100 units/mL penicillin/streptomycin/gentamicin at 37 degree as described.Hypothalamic tissue removal and primary hypothalamic neurons isolation and culture were basically described previously2.6pm, 3 times per week for three weeks. For the effect of TNF‐α on sleep, mice  were studied by video tracking similarly as describedam and off at 5 pm). All animals were identified by earmarks and numbered accordingly. Using a table of random numbers, animals were randomly divided into experimental groups. Animals from experimental groups were always treated and assessed first, followed by control groups. During experiments and analysis, the investigators were blinded to genotype and experimental group. Animals were identified by earmarks and got numbers, which were announced to the investigator only after finishing experiments and analysis. Data were recorded and analysed by sleep sign system according to the criteria described previously.The research has been approved by local Animal Care and Use Committee (IACUC #09243 and #21589). For the effect of TNF‐α on hypocretin expression in hypothalamus, one‐time intraperitoneal injections of TNF‐α at indicated dose or control vehicle saline were performed. TNF‐α levels in serum were examined 1 hour after injection. TNF‐α levels in hypothalamic tissues were examined at 6 hour after injection. For neutralizing studies, mice received intravenous injection of rat TNF‐α neutralizing antibody (1 mg/kg whole body weight) or control rat IgG 30 minutes before TNF‐α injection. For the effect of blocking hypocretin signalling, the HcrtR1/OX1R antagonist SB334867 was used. SB334867 was dissolved in dimethyl sulphoxide (DMSO) and diluted in sterile saline (30 mg/kg) for intraperitoneal administration 30 minutes before TNF‐α injection. For the effects of TNF‐α on sleep, muscle activity, learning, cognition, memory, intraperitoneal injection of TNF‐α or control saline were performed at 5 For the effect of TNF‐α on learning, cognition, and memory, spatial reference learning and memory abilities were assessed in a modified open field Morris water maze basically as described previously.Novel object recognition (NOR) model is widely used as a simple behavioural assay for the investigation of alterations in recognition memory. To examine the effect of TNF‐α on NOR, the analysis was performed basically as described previously.2.7The research has been approved by local Institutional Review Board (IRB #N13106548), and informed consent was obtained. The retrospective analysis of RBD frequency was based on using unidentified records of questionnaire, which is a widely used and well‐established single question questionnaire with a sensitivity of 94% and a specificity of 87% for RBD.2.8t test analysis (for comparisons between two groups) . Data points outside the 95% confidence interval were treated as outliers and excluded from the data analysis. A P value of <0.05 was considered statistically significant.Data were analysed using the unpaired or paired 33.1P < 0.01). TNF‐α treatment simultaneously decreased hypocretin 2 protein levels in a dose‐dependent manner when compared to that in controls .We have previously reported that TNF‐α decreased hypocretin expression in rat B35 neuroblastoma cells in which exogenous hypocretin was overexpressed mediated by retroviral vectors.3.2Prepro‐hypocretin is the single precursor of both hypocretin 1 and hypocretin 2; prepro‐hypocretin generates hypocretin 1 and hypocretin 2 through proteolytic cleavage.3.3Pre‐treatment of TNF‐α neutralizing antibody blocked TNF‐α‐induced reduction of prepro‐hypocretin protein levels in primary hypothalamic neurons Figure A and B. 3.4To examine the effects of TNF‐α on prepro‐hypocretin expression in vivo in hypothalamus, TNF‐α or control saline solution was intraperitoneally injected Figure . Serum T3.5TNF‐α significantly decreases the expression of hypocretin 1 and hypocretin 2 in hypothalamus in mice Figure . To exam3.6Hypocretin (aka orexin)‐deficient mice show sleep fragmentation with more transitions between wake, non‐rapid eye movement (NREM) and rapid eye movement (REM) sleeps.It has been reported that microinjection of hypocretin in the locus coeruleus and pontine inhibitory area results in muscle tone changes.3.7Evidence is accumulated to support the involvement of neuroinflammation in neurodegenerative diseases, including Alzheimer's and Parkinson's diseases.4Hypocretin 1 and hypocretin 2 (aka orexin A and B) have critical roles in the regulation of sleep, wakefulness, feeding behaviour and emotion.TNF‐α is involved in the regulation of sleep‐wakefulness behaviour and fatigue during infections and depression.The critical role of hypocretin system in sleep disorders is learned from studies in narcolepsy patients and in knockout mice.Our study examined the effects of relatively long‐term or chronic TNF‐α treatment Figure . It is pMicroinjection of hypocretin in the locus coeruleus and pontine inhibitory area results in muscle tone changes,In conclusion, our work provides evidence that the hypocretin system can be impaired by TNF‐α directly in vivo in mice. The data provide evidence and mechanism that TNF‐α functions as a negative regulator of the hypocretin system in vivo. In addition, repeated TNF‐α challenge induces RBD‐like behaviour and sleep dysfunction, and decreases learning, cognition and memory in mice. This new evidence shed some lights on the pathophysiological role of chronically elevated TNF‐α. Our findings add a new dimension to the pathophysiological role of TNF‐α along with an impaired hypocretin system that may have important implications in health care and neurodegenerative diseases.The authors confirm that there are no conflicts of interests.SZ, PC, XH and QD conceived and designed this study; PC, XKZ, SL, NL, YD, JL, KD, LH and MH performed the research; SZ, PC, XH and QD analysed the data; SZ, NL, YD, JL, ZH, LW, YW, XH and QD contributed materials; and SZ, PC, XH and QD wrote the paper. All authors read and approved the content.


A new highly efficient three-component reaction of alkyl acrylate, aldehyde and dialkyl malonate using ethyl diphenylphosphine as organocatalyst has been described. Various highly functional compounds bearing hydroxyl groups and the ester functions can be easily prepared in moderate to good yields according to our one-step procedure. The reactions are believed to proceed via Morita-Baylis-Hillman reactions of alkyl acrylate and aldehydes, followed by the Michael addition reactions of dialkyl malonates. Our reactions indicated that the intermediate species formed in the phosphine-catalyzed MBH reaction are an effective organic base to catalyze the Michael addition reactions of dialkyl malonates to the preformed MBH adducts. Carbon-carbon bond formation is the most important organic reaction because it plays a fundamental role in making carbon-frameworks of organic compounds for numerous interesting studies concerning reactivity, chemoselectivity, and stereoselectivity ,2. MultiThe Baylis-Hillman reaction adduct, resulting from alkyl acrylate and aldehyde is a good Michael acceptor because of the activated ester functionality by the neighboring hydroxy group ,18,19,201 and alkyl acrylate 2, which is followed by the Michael addition of dialkylmalonate 3 to the resulting adduct +, 380 (8), 319 (80), 302 (77), 274 (43), 245 (72), 160 (100), 133 (82), 127 (85), 113 (48), 105 (52), 55 (81); HRMS (MALDI) for C18H23NO9Na, [M+Na]+ (420.1270) found: 420.1275.Threo-1,1-diethyl 3-methyl 4-hydroxy-4-(4-nitrophenyl)butane-1,1,3-tricarboxylate (threo-4a). 1H-NMR  δ/ppm: 8.22 , 7.50 , 4.98 , 4.23–4.14 , 3.64 , 3.38 , 2.97–2.90 , 2.30–2.21 , 2.18–2.09 , 1.29–1.21 ; 13C-NMR  δ/ppm: 173.8, 168.6, 168.5, 148.7, 147.6, 127.1, 123.7, 73.8, 61.8, 61.7, 52.1, 49.8, 49.6, 27.9, 14.0; MS  m/z (%): 398 (1) [M+1]+, 380 (5), 319 (85), 302 (79), 274 (40), 245 (77), 223 (86), 160 (100), 133 (93), 127 (85), 113 (51), 105 (49), 55 (81); HRMS (MALDI) for C18H23NO9Na, [M+Na]+ (420.1270) found: 420.1283.Erythro-1,1-diethyl 3-methyl 4-hydroxy-4-(3-nitrophenyl)butane-1,1,3-tricarboxylate (erythro-4b). 1H-NMR  δ/ppm: 8.23 , 8.17–8.11 , 7.69 , 7.52 , 5.14 , 4.22–4.06 , 3.65 , 3.37 , 2.90–2.82 , 2.35–2.25 , 2.18–2.07 , 1.28–1.14 . 13C-NMR  δ/ppm: 173.9, 168.9, 168.7, 148.3, 143.2, 132.2, 129.3, 122.8, 121.2, 72.7, 61.7, 61.6, 52.2, 49.9, 49.8, 25.4, 13.9. MS  m/z (%): 398 (5) [M+1]+, 380 (10), 348 (5), 246 (41), 214 (11), 187 (11), 173 (7), 160 (100), 134 (20). HRMS (ESI) for C18H23NO9Na, [M+Na]+ (420.1270) found: 420.1268.Threo-1,1-diethyl 3-methyl 4-hydroxy-4-(3-nitrophenyl)butane-1,1,3-tricarboxylate (threo-4b). 1H-NMR  δ/ppm: 8.16 , 8.15–8.08 , 7.65 , 7.51 , 4.95 , 4.18–4.07 , 3.63 , 3.60 , 3.33 , 2.95–2.85 , 2.24–2.12 , 2.08–1.98 , 1.25–1.15 . 13C-NMR  δ/ppm: 173.7, 168.6, 168.4, 148.2, 143.6, 132.3, 129.4, 122.9, 121.2, 73.7, 61.7, 61.6, 52.0, 50.0, 49.6, 27.7, 13.9, 13.7. MS  m/z (%):398 (1) [M+1]+, 380 (5), 348 (5), 301 (6), 246 (41), 214 (8), 187 (11), 173 (8), 160 (100), 134 (5). HRMS (ESI) for C18H23NO9Na, [M+Na]+ (420.1270) found: 420.1273.Erythro-1,1-diethyl 3-methyl 4-hydroxy-4-(2-nitrophenyl)butane-1,1,3-tricarboxylat (erythro-4c). 1H-NMR  δ/ppm: 8.00 , 7.89 , 7.68 , 7.47 , 5.72 , 4.19–4.10 , 4.07 , 3.69 , 3.43 , 3.36 , 2.98 , 2.38–2.28 , 2.17–2.08 , 1.23 , 1.15 . 13C-NMR  δ/ppm: 174.6, 168.9, 168.6, 147.5, 136.1, 133.5, 129.3, 128.7, 124.9, 69.2, 61.6, 61.5, 52.2, 49.9, 47.6, 25.1, 13.9. MS  m/z (%): 398 (100) [M+1]+, 385 (62), 380 (61), 363 (75). HRMS (FAB) for C18H24NO9, [M+H]+ (398.1451) found: 398.1457.Threo-1,1-diethyl 3-methyl 4-hydroxy-4-(2-nitrophenyl)butane-1,1,3-tricarboxylate (threo-4c). 1H-NMR  δ/ppm: 7.98 , 7.71–7.61 , 7.49–7.42 , 5.46 , 4.25–4.14 , 3.84 , 3.58 , 3.43 , 3.11–3.04 , 2.45–2.34 , 2.30–2.20 , 1.30–1.22 . 13C-NMR  δ/ppm: 174.3, 168.9, 168.8, 148.1, 137.4, 133.7, 129.0, 128.6, 125.1, 70.2, 61.9, 52.1, 49.9, 48.9, 28.8, 14.1. MS (FAB) m/z (%): 398 (100) [M+1]+, 385 (60), 380 (65), 363 (72). HRMS (FAB) for C18H24NO9, [M+H]+ (398.1451) found: 398.1454.Erythro-1,1-diethyl 3-methyl 4-hydroxy-4-(4-(trifluoromethyl)phenyl)butane-1,1,3-tricarboxylate (erythro-4d). 1H-NMR  δ/ppm: 7.60 , 7.47 , 5.10 , 4.19–4.03 , 3.64 , 3.36 , 3.17 , 2.88–2.79 , 2.35–2.24 , 2.19–2.09 , 1.22 , 1.17 . 13C-NMR . δ/ppm: 174.1, 168.9, 168.7, 144.9, 130.0 , 126.7, 125.2 , 123.9 , 130.0 , 126.4, 125.3 , 123.8 , 73.1, 61.6, 61.5, 52.1, 49.9, 49.8, 25.4, 14.0, 13.9. MS (FAB) m/z (%): 420 (1) [M]+, 403 (10), 360 (58), 245 (42), 173 (31), 160 (100), 126 (38), 55 (20). HRMS (ESI) for C19H23F3NaO7, [M+Na]+ (443.1294) found: 400.1290.Threo-1,1-diethyl 3-methyl 4-hydroxy-4-(4-(trifluoromethyl)phenyl)butane-1,1,3-tricarboxylate (threo-4d). 1H-NMR  δ/ppm: 7.57 , 7.40 , 4.85 , 4.15–4.07 , 3.61 , 3.30 , 2.90–2.81 , 2.19–2.08 , 2.01–1.90 , 1.24–1.13 . 13C-NMR  δ/ppm: 73.8, 168.5, 168.4, 145.3, 130.0 , 126.7, 125.2 , 123.9 , 74.2, 61.5, 61.4, 52.7, 50.2, 49.6, 27.5, 13.6. MS  m/z (%): 420 (3) [M]+, 403 (14), 360 (69), 245 (34), 173 (27), 160 (100), 126 (42), 55 (20). HRMS (ESI) for C19H23F3NaO7, [M+Na]+ (443.1294) found: 400.1288.Erythro-1,1-diethyl 3-methyl 4-(4-cyanophenyl)-4-hydroxybutane-1,1,3-tricarboxylate (erythro-4e). 1H-NMR  δ/ppm: 7.65 , 7.48 , 5.10 , 4.20–4.07 , 3.66 , 3.36 , 3.20 , 2.88–2.80 , 2.34–2.24 , 2.15–2.05 , 1.28–1.16 . 13C-NMR  δ/ppm: 174.0, 168.9, 168.7, 146.2, 132.2, 126.7, 118.6, 111.7, 73.0, 61.7, 61.6, 52.2, 49.8, 49.7, 25.3, 14.0. HRMS (MALDI) for C19H23NNaO7, [M+Na]+ (400.1372) found: 400.1385.Threo-1,1-diethyl 3-methyl 4-(4-cyanophenyl)-4-hydroxybutane-1,1,3-tricarboxylate (threo-4e). 1H-NMR  δ/ppm: 7.61 , 7.41 , 4.87 , 4.18–4.07 , 3.60 , 3.55 , 3.30 , 2.89–2.80 , 2.20–2.10 , 2.03–1.91 , 1.25–1.16 . 13C-NMR  δ/ppm: 173.7, 168.5, 168.4, 146.7, 132.2, 127.0, 118.5, 111.7, 74.0, 61.7, 61.6, 52.0, 49.9, 49.5, 27.7, 13.9. MS  m/z (%):78 (14) [M+1]+, 360 (13), 317 (39), 300 (29), 282 (16), 246 (21), 203 (31), 186 (29), 160 (100), 133 (15). HRMS (MALDI) for C19H23NNaO7, [M+Na]+ (400.1372) found: 400.1385.1,1-Diethyl 3-methyl 4-hydroxy-4-(pyridin-3-yl)butane-1,1,3-tricarboxylate (4f). 1H-NMR  δ/ppm: 8.44 , 7.71–7.61 , 7.50–7.40 , 7.27–7.22 , 4.99 and 4.85 , 4.42 , 4.19–4.04 , 3.65 and 3.55 , 3.44–3.22 , 2.95–2.74 , 2.36–1.86 , 1.29–1.12 . 13C-NMR  δ/ppm: 173.8, 173.5, 168.9, 168.7, 168.6, 168.4, 149.2, 148.1, 137.1, 134.2, 134.1, 131.6, 130.8, 130.7, 128.6, 128.5, 123.5, 72.7, 71.6, 61.6, 61.5, 61.4, 51.9, 51.8, 50.4, 50.2, 49.9, 49.7, 27.7, 26.0, 13.9.Threo-triethyl 4-hydroxy-4-(4-nitrophenyl)butane-1,1,3-tricarboxylate (threo-4g). 1H-NMR  δ/ppm: 8.16 , 7.48 , 4.93 , 4.20–4.09 , 4.09–4.01 , 3.68 , 3.33 , 2.90–2.81 , 2.24–2.13 , 2.09–2.00 , 1.21 , 1.12 . 13C-NMR  δ/ppm: 173.3, 168.6, 168.4, 148.8, 147.4, 127.1, 123.5, 73.6, 61.7, 61.6, 61.2, 49.8, 49.5, 27.9, 13.9, 13.8. MS (MALDI) m/z (%): 434 (100) [M+Na]+, 368 (55), 352 (63). HRMS (MALDI) for C19H25NNaO9, [M+Na]+ (434.1427) found: 434.1436.Threo-triethyl 4-hydroxy-4-(3-nitrophenyl)butane-1,1,3-tricarboxylate (threo-4h). 1H-NMR  δ/ppm: 8.18, , 8.15–8.09 , 7.66 , 7.52, , 4.96 , 4.21–4.12 , 4.09 , 3.60 , 3.37 , 2.95–2.84 , 2.28–2.18 , 2.14–2.03 , 1.28–1.19 , 1.14 . 13C-NMR  δ/ppm: 173.3, 168.6, 168.5, 148.3, 143.7, 132.3, 129.4, 122.9, 121.2, 73.7, 61.7, 61.6, 61.2, 49.8, 49.6, 27.9, 13.9. MS  m/z (%):412 (4) [M+1]+, 393 (5), 320 (14), 302 (15), 260 (25), 185 (39), 160 (100), 133 (15), 55 (16). HRMS (MALDI) for C19H25NNaO9, [M+Na]+ (434.1427) found: 434.1431.Threo-triethyl 4-(4-cyanophenyl)-4-hydroxybutane-1,1,3-tricarboxylate (threo-4i). 1H-NMR  δ/ppm: 7.65 , 7.45 , 4.91 , 4.25–4.14 , 4.09 , 3.38 , 3.34 , 2.93–2.83 , 2.31–2.20 , 2.19–2.07 , 1.26 , 1.15 . 13C-NMR  δ/ppm: 173.1, 168.5, 168.3, 146.8, 132.0, 126.9, 118.4, 111.5, 73.9, 61.5, 61.4, 61.0, 49.8, 49.4, 27.7, 13.8. MS  m/z (%): 391 (4) [M]+, 218 (19), 160 (100), 132 (10), 102 (30). HRMS (MALDI) for C20H25NNaO7, [M+Na]+ (414.1529) found: 414.1535.Threo-ethyl 3-(4-cyanophenyl)-2-methyl)-3-hydroxypropanoate (threo-4j). 1H-NMR  δ/ppm: 8.35 , 7.65 , 7.22 , 4.81 , 4.16–3.96 , 3.26 , 2.85–2.74 , 2.14–2.01 , 1.94–1.82 , 1.22–1.09 . 13C-NMR  δ/ppm: 173.2, 168.5, 168.4, 148.7, 147.8, 137.3, 134.1, 123.5, 72.5, 61.5, 61.4, 60.9, 50.2, 49.6, 27.7, 13.9, 13.8, 13.7. MS (MALDI) m/z (%): 368 (100) [M+1]+, 350 (38), 305 (50), 289 (37). HRMS (MALDI) for C18H26NO7, [M+H]+ (368.1709) found: 368.1719.Threo-trimethyl 4-hydroxy-4-(4-nitrophenyl)butane-1,1,3-tricarboxylate (threo-4k). 1H-NMR  δ/ppm: 8.20 , 7.50 , 4.96 , 3.71 , 3.65 , 3.41 , 2.92–2.88 , 2.24–2.19 , 2.13–2.08 . 13C-NMR  δ/ppm: 173.6, 168.9, 168.6, 148.5, 147.6, 127.1, 123.7, 73.9, 52.8, 52.7, 52.1, 49.8, 49.2, 27.9. MS  m/z (%): 369 (8) [M]+, 317 (39), 300 (29), 282 (16), 246 (21), 152 (31), 145 (29), 131 (100), 86 (46). HRMS (MALDI) for C16H19NNaO9, [M+Na]+ (392.0957) found: 369.0968.Erythro-methyl 2-(hydroxy(4-nitrophenyl)methyl)-4,4-bis(phenylsulfonyl)butanoate (erythro-5). mp.: 96.1–96.5 °C.1H-NMR  δ/ppm: 8.21 , 7.88 , 7.74–7.60 , 7.60–7.49 , 7.45 , 5.26 , 4.87 , 3.60 , 3.41–3.31 , 3.12 , 2.72–2.60 , 2.31–2.19 . 13C-NMR  δ/ppm: 172.7, 147.9, 147.5, 137.4, 136.8, 134.7, 129.6, 129.4, 129.2, 129.0, 127.2, 123.6, 80.6, 72.5, 52.5, 49.7, 22.5. HRMS (ESI) for C24H23NNaO9S2, [M+Na]+ (556.0712) found: 556.0717. CCDC number: 837000.Threo-methyl 2-(hydroxy(4-nitrophenyl)methyl)-4,4-bis(phenylsulfonyl)butanoate (threo-5). mp.: 140.2–140.7 °C. 1H-NMR  δ/ppm: 8.18 , 7.86 , 7.81 , 7.69 , 7.60–7.49 , 7.47, 5.07 , 4.73 , 3.57 , 3.54–3.45 , 3.36 , 2.54–2.42 , 2.38–2.26 . 13C-NMR  δ/ppm: 172.4, 147.8, 147.6, 137.2, 137.1, 134.8, 134.7, 129.6, 129.5, 129.2, 129.1, 127.2, 123.7, 80.6, 73.4, 52.4, 49.9, 24.8. HRMS (ESI) for C24H23NNaO9S2, [M+Na]+ (556.0712) found: 556.0710.2,2-Diethyl 4-methyl 5-hydroxy-5-(4-nitrophenyl)pentane-2,2,4-tricarboxylate (6). 1H-NMR  δ/ppm: 8.18 , 7.47 , 4.99 , 4.17–4.11 , 3.56 , 2.98–2.97 , 2.31 , 2.15 , 1.36 , 1.24–1.17 . 13C-NMR  δ/ppm: 174.0, 171.9, 171.5, 148.8, 147.5, 127.0, 123.5, 74.8, 61.7, 61.6, 52.9, 51.9, 48.6, 34.3, 20.2, 13.9, 13.8. MS (ESI) m/z (%): 434 (100) [M+Na]+, 305 (5). HRMS (MALDI) for C19H25NNaO9, [M+Na]+ (434.1427) found: 434.1438.in-situ generated MBH adducts. This multicomponent reaction has a broad reaction scope with all three components of acrylate, aldehyde and activated alkane. Further mechanistic details and development of their asymmetric three-component reactions, are now underway in our laboratory.In summary, we have developed a novel, simple approach toward the synthesis of highly functionalized molecules via three-component reactions of substituted aromatic aldehydes, alkyl acrylates and activated alkane catalyzed by ethyl diphenylphosphine. Furthermore, we presented the first organocatalytic addition of carbon-nucleophile to the


The recent discovery of the Lokiarchaeota and other members of the Asgard superphylum suggests that closer analysis of the cell biology and evolution of these groups may help shed light on the origin of the eukaryote cell. Asgard lineages often appear in molecular phylogenies as closely related to eukaryotes, and possess “Eukaryote Signature Proteins” coded by genes previously thought to be unique to eukaryotes. This phylogenetic affinity to eukaryotes has been widely interpreted as indicating that Asgard lineages are “eukaryote-like archaea,” with eukaryotes evolving from within a paraphyletic Archaea. Guided by the established principles of systematics, we examine the potential implications of the monophyly of Asgard lineages and Eukarya. We show that a helpful parallel case is that of Synapsida, a group that includes modern mammals and their more “reptile-like” ancestors, united by shared derived characters that evolved in their common ancestor. While this group contains extinct members that share many similarities with modern reptiles and their extinct relatives, they are evolutionarily distinct from Sauropsida, the group which includes modern birds, reptiles, and all other amniotes. Similarly, Asgard lineages and eukaryotes are united by shared derived characters to the exclusion of all other groups. Consequently, the Asgard group is not only highly informative for our understanding of eukaryogenesis, but may be better understood as being early diverging members of a broader group including eukaryotes, for which we propose the name “Eukaryomorpha.” Significantly, this means that the relationship between Eukarya and Asgard lineages cannot, on its own, resolve the debate over 2 vs. 3 Domains of life; instead, resolving this debate depends upon identifying the root of Archaea with respect to Bacteria. The discovery of Archaea  has had There is no doubt that the eukaryotes descend from a common ancestor , as there are many characters that trace back to LECA, uniting modern eukaryotes as a group. These characters include the mitochondrion, nuclear envelope, nuclear pores and an endomembrane system, linear chromosomes, spliceosomal machinery and introns, among many others  are symplesiomorphies, having evolved well before eukaryotes. We contend that ESPs that are shared with Asgard lineages, but not with other Archaea, are best interpreted as synapomorphies if the sister relationship between Asgard and Eukarya is correct, and that these together form a natural monophyletic group to the exclusion of other Archaea. Importantly, this does not make Asgard lineages eukaryotes; there is no evidence (as yet) that these lineages carry nuclei or other endomembrane systems associated with the eukaryote cell. Moreover, it does not indicate that Asgard lineages are Figure 1). The deepest split in the evolutionary history of amniotes is that between the groups Sauropsida and Synapsida, congruent to the split between extant reptiles and mammals. After this deep divergence, Sauropsida diversified into groups including all extant and extinct reptile lineages, including birds. Synapsida diversified into many lineages as well. Of these, only mammals survived, although there is a rich, diverse evolutionary history of extinct mammalian relatives and ancestors . Nevertheless, the presence of some shared derived characters, including a single temporal fenestra and slightly enlarged canines identify this extinct group as members of Synapsida , one would expect the earliest relatives of the ancestors of modern eukaryotes to resemble Archaea, with perhaps only a few derived characters in common with extant eukaryotes. In such a case, while the majority of characters would be ancestral and archaeal-like, under a systematics framework and a clade-based definition of Archaea, these characters (symplesiomorphies) do not, on their own, bestow status as Archaea to the exclusion of Eukarya. Rather, being more closely related to Eukarya than the archaeal outgroup, and being united by derived characters shared with Eukarya, they would be properly identified as belonging to a clade including Eukarya. Unlike The phylogenetic placement of eukaryotes with relation to Archaea is a different question than the process of the origin of the eukaryote-type cell. These questions are related by the eukaryal stem, the lineage along which all eukaryote-specific characters were acquired during perhaps over a billion years of evolution. The deeper the stem ancestor, the fewer derived characters will be present. It directly follows that, immediately following the divergence of the eukaryal stem lineage from its outgroup, one would expect few, if any derived characters, and the preservation of most, if not all ancestral characters. An abundance of ancestral characters present within a deeply branching lineage is thus a poor rationale for its placement with the outgroup, rather than with a more highly derived sister group. This observation is important in establishing principles for accurately describing the ancestry of eukaryotes.extant lineages more closely related to Eukarya than Archaea . Synapomorphy-based approaches  are necessary to provide this distinction.Using a clade-based approach, defining eukaryotes is trivial – they are all descendants of the last common ancestor of extant Eukarya, i.e., “crown Eukarya.” All extinct stem lineage members then belong to broader groupings inclusive of crown Eukarya, based on nested subsets of synapomorphies, as is the case with increasingly mammal-like groups nested within the synapsid lineage leading to crown group mammals . AlternFigure 2). The analogy here is to Sauropsida and Synapsida in Figure 1, where reptile-like forms are present on both sides of tree, but are not diagnostic of clade membership. While this rooting has not been recovered so far by any major published phylogenetic analyses including Archaea, Asgard, and Eukarya, the rooting of the archaeal tree remains contentious, and is highly sensitive to evolutionary and phylogenetic reconstruction models, taxon sampling, protein datasets, and alignment site sampling  would exclude Asgard from Archaea, and remain entirely consistent with a 3-Domain Tree of Life, with Asgard included within a broader sister clade also including Eukarya, and of equal taxonomic rank to Archaea  present across Euryarchaeota, DPANN, and TACK clades, but absent in Asgard and Eukarya. Complete genome sequencing of members of the Asgard superphylum will allow this prediction to be tested.Figure 3).What if Archaea + Eukarya are rooted on a different branch, so that a monophyletic Eukarya + Asgard groups within extant archaeal diversity? As a consequence of this rooting, Archaea would in fact be paraphyletic, consistent with a “2-Domain” hypothesis for the Tree of Life. How then, would Eukarya’s relationship to Asgard be best understood? If Eukarya roots within extant archeal diversity, one can more clearly polarize the shared ancestral characters within Eukarya, and infer that the earliest stem eukaryotes were essentially archaeal, in both a systematic and physiological sense. However, the same shared derived characters of ESPs would still unite Eukarya and Asgard, and their monophyly would still stand, albeit as a clade of a lower taxonomic rank than Archaea  and other reptilian lineages. The extant sister group to birds, Crocodilia, retains several ancestral characters in common with other reptile outgroups, but also show derived characters linking them to birds within the wider group Archosauria, including their extinct relatives, pterosaurs and non-avian dinosaurs   . This plOne derived character often elevated to be the defining trait of “true” Eukarya is the presence of mitochondria, to the extent that it has been proposed that Eukarya are the product of a “merger” of a bacterial and archaeal lineage, and that this event triggered the evolution of all additional Eukarya-specific characters, such as introns, the nucleus, and endomembrane system . Should The mitochondrial acquisition represents three specific changes to the eukaryal ancestor lineage, metabolic (the acquisition of aerobic respiration), genetic , and cytological (the maintenance of a highly derived replicating organelle). The different aspects of this single event can, conceptually, be considered individually.If there had never been an endosymbiotic event giving rise to mitochondria, but the genes for aerobic respiration had nevertheless been acquired by horizontal gene transfer (HGT) from an alphaproteobacterial lineage, would this event, in itself, have the same weight as a defining character for Eukarya? Metabolic innovations, including aerobic respiration, often evolve via HGT across microbial lineages. The widespread distribution of aerobic respiration across the Tree of Life shows that this character would be a poor synapomorphy to define Eukarya as a distinct Domain of life. From a genetic perspective, many genes of bacterial origin were transferred as a consequence of the mitochondrial endosymbiotic event, although it is likely that many of the genes also shared between Eukarya and Bacteria were not necessarily acquired in this way, as they have evidence of different evolutionary histories . FurtherFigure 4).The acquisition of mitochondria is therefore most unique and distinct from archaeal and bacterial evolutionary processes from a cytological perspective. Obligate, co-evolved endosymbioses involving radical genome reduction are common among bacterial endosymbionts of eukaryotes . In contGene tree phylogenies tend to recover the monophyly of Eukarya and Asgard groups. ESPs have been identified in these newly discovered groups, reinforcing this proposed relationship. We propose that these results are consistent with the interpretation that Eukarya and Asgard lineages form a distinct clade defined by shared derived characters. Following the divergence of Asgard and Eukarya, stem eukaryotes continued to acquire a large number of derived physiological characters, including mitochondria, that would come to represent the “modern” eukaryotic cell. A correct application of cladistics requires grouping based on shared derived characters. Many ancestral archaeal-like characters were retained in the Asgard lineages, and these would have been present in the earliest direct stem Eukarya ancestors, as well. These ancestral characters are, in themselves, insufficient to define Asgard lineages as members of Archaea except in the sense that, under 2-Domain tree topologies, both Asgard and Eukarya are part of the archaeal tree. Therefore, a monophyletic relationship between Eukarya and Asgard cannot be used to distinguish between traditional “2-Domain” and “3-Domain” hypotheses for the Tree of Life. Rather, the placement of the root leading to Bacteria provides this distinction. While current phylogenomic analyses favor a rooting within Archaea, this placement is highly sensitive to a variety of factors, and continues to be debated. Both 2-Domain and 3-Domain scenarios are compatible with treating Asgard lineages as members of a distinct and broader group including eukaryotes that requires taxonomic recognition.GF originated and devised the study, which was further developed in collaboration with AP. GF drafted the figures. GF and AP drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N = 9), genome-wide SNP arrays (N = 418), whole-exome sequencing (N = 26), and whole-genome sequencing (N = 10). Comprehensive analysis did not identify plausible variants of strong effect. Rather, pedigree cases had significantly higher genetic risk scores compared to pedigree and community controls.We searched for genetic causes of major psychiatric disorders  in a large, densely affected pedigree from Northern Sweden that originated with three pairs of founders born around 1650. We applied a systematic genomic approach to the pedigree via karyotyping ( Most of the implicated findings to date have been for common genetic variants4, with significant common-variant genetic correlations between these disorders5. Although these results are unquestionably informative6, they usually implicate broad genomic regions with imprecise connections to particular genes. This complicates the design of subsequent neuroscience experiments of which major tools are optimal for the evaluation of single genes.The genetic basis of major psychiatric disorders  has been intensively investigated over the past decade atop of substantial efforts dating to 1980s1. An optimal strategy would apply assays that cover the most likely types of genetic variation coupled with control of multiple comparisons to enhance reproducibility. Although dense pedigrees can result from high common-variant genetic burden7, they can also be explained by rare variation of strong effect inherited from common ancestors. Systematic searches of such pedigrees are a complementary strategy to attempt to identify genetic variants of strong effect.An experimental design that has been insufficiently exploited in psychiatry genetics in the current era is the comprehensive study of large, densely affected pedigrees8 with a regional prevalence of major psychiatric disorders around eight times higher than Sweden as a whole9. There is a large pedigree in Northern Sweden of which origins (based on contemporaneous church records) date to three founder couples born around 1650. Individuals in this 14-generation pedigree  in a subset of the pedigree.Northern Sweden has a notably high prevalence of bipolar disorder and schizophreniaree Fig.  have incN = 9), genome-wide SNP arrays (N = 418), whole-exome sequencing (N = 26), and whole-genome sequencing (N = 10). Our goal was to identify a genetic contribution to the unusually high prevalence of major psychiatric disorders in this multiplex pedigree. Potential explanations range from a genetic variant of strong effect inherited from a common ancestor  to an unusually high burden of common variants that individually have weak effects . The latter possibility has been suggested by theory10 and demonstrated empirically for bipolar disorder7.We applied a systematic genomic approach to the pedigree. Genomic assays included karyotyping . All participants provided written informed consent for participation in the study.This Northern Swedish pedigree Fig.  is from Table Karyotyping was performed on fresh venous blood samples using standard methods in a hospital-based clinical genetics unit. The goal was to evaluate “microscopic” structural variants types of which can be missed by other methods.Microsatellite markers: For linkage analysis, 472 short tandem repeat markers were genotyped , 411 markers with a mean inter-marker distance of 7.6 cM, and 61 additional markers for fine-mapping in eight chromosomal regions. PCR amplifications were performed under standard reaction conditions using fluorescent-labeled primers. PCR-products were sized on an ABI 3730 Sequencer (Applied Biosystems) and genotypes were scored using TracI. Subjects included affected individuals  and their unaffected first-degree relatives (N = 226). Linkage regions were used to prioritize genomic regions for evaluation.SNP genotyping was done using OmniExpress arrays , and genotypes were called using BeadStudio (Illumina). Quality control filters excluded SNPs that had missingness ≥0.05, MAF <0.01, >2 Mendelian errors in trios, or violated Hardy-Weinberg Equilibrium (PHWE < 1 x 10−6) in 201 unaffected individuals from the pedigree. A total of 621,639 SNPs passed quality control. Subject quality control filters excluded one subject with missingness >0.03, and one member in each of two monozygotic twin pairs were removed. These data were used to identify large structural variants, to estimate GRS, and to prioritize genomic regions for evaluation.Array-based structural variation: As detailed elsewhere13, we evaluated autosomal structural variation using PennCNV14, and removed lower confidence calls  or structural variants mostly overlapping a gap in the reference genome or a region subject to recombination in peripheral white blood cells. One sample with aberrant log-R ratio was removed. Neighboring structural variants were merged. Case-control studies often focus on uncommon or rare structural variation . We did not apply this filter to this pedigree as not to exclude a potentially salient structural variant carried by multiple cases.Whole-exome sequencing (WES) used exome capture , followed by WES using Illumina HiSeq 2000 and HiSeq 2500 instruments . We used the Genome Analysis Toolkit (GATK) Version 2 Best Practices pipeline for preprocessing and variant discovery. Key steps for preprocessing included read alignment to hg19 using bwa-mem, removal of duplicated reads, local realignment around indels, and base quality recalibration, which result in analysis-ready .bam files. Key steps for variant discovery included variant calling using HaplotypeCaller (per-sample) to generate single-sample gVCFs, joint genotyping calling on the gVCFs for all samples, and variant recalibration to assign a well-calibrated probability to each variant call. The result is a VCF file for all samples, based on which we retained only the PASS variants and separated SNPs from indels. Quality control of the .bam files was done using Picard tools to evaluate the distributions of quality metrics. Quality control of VCF files was done using the “CollectVariantCallMetrics” tool in Picard to evaluate the distribution of SNV quality metrics. SNP genotype concordance was >0.988 for all samples based on 23,783 overlapping SNPs between WES and OmniExpress arrays, indicating high reliability for PASS SNP calls. These data were used to evaluate whether rare, protein-altering exon variants were plausibly causal in the pedigree.Whole-genome sequencing (WGS): We selected 10 affected individuals with bipolar disorder from across the pedigree for WGS. These subjects did not carry a large CNV and were more distantly related . These data were used as a means to identify types of variation that would have been missed by other technologies.16 under an affected-only model with unaffected relatives considered “unknown”. Linkage results were converted to hg19 coordinates by mapping microsatellite probe sequences. Second, we applied BEAGLE 17 to genome-wide SNP data to estimate pairwise IBD segments in all genotyped individuals (broad affection definition versus controls and narrow versus controls). We applied DASH 18 to the pairwise IBD segments to infer haplotype clusters of IBD individuals. Due to the relatedness of subjects in the pedigree, we used GCTA (v1.26.0)20 to perform linear mixed model association testing. GCTA analyses had case-control status as the dependent variable, haplotype clusters as markers, and included the genetic relationship matrix estimated from autosomal genome-wide SNPs.We searched for genomic regions inherited IBD from a common ancestor. First, we used genetic linkage analysis based on 472 polymorphic short tandem repeat markers genotyped in 152 narrow-affected and 226 unaffected individuals . As linkage analyses on the complete pedigree were intractable, the pedigree was split in sub-pedigrees (maximum 400 bits). Multipoint nonparametric and parametric linkage analysis were done using Simwalk2 (v2.91)21 or schizophrenia22 using partitioned LD score regression (pLDSC)24. pLDSC is an extension of LD score regression allowing estimation of whether a set of genomic regions  are enriched for the SNP-heritability of a trait based on GWAS summary statistics. We followed the authors’ recommendations in order to add our regions of interest to the pLDSC baseline model23. Significance was assessed using the enrichment P-value.We evaluated whether selected genomic regions could be linked to GWAS results for bipolar disorder21 and schizophrenia22 using best practice procedures26. We contrasted cases and controls from the pedigree with a population-based control sample from the same region of Sweden22. All three groups were genotyped with Illumina OmniExpress arrays. To create training sets, we obtained GWAS summary statistics (URLs) and used METAL27 to redo meta-analysis of case-control data from European samples after excluding Swedish studies26. These training sets were used to select SNPs and weight risk alleles for GRS calculation . To select relatively independent SNPs for GRS calculation, we applied LD-clumping (r2 < 0.1 in 1 Mb windows) using 1000 Genomes Project European samples as the LD reference.We computed GRS for bipolar disorderN = 81), schizoaffective disorder (N = 29), schizophrenia (N = 18), bipolar 2 disorder (N = 67), bipolar disorder NOS (N = 22), or no lifetime history of a serious psychiatric disorder (N = 201). There were 244 females (58.4%) and 174 males (41.6%). The narrow definition of illness included bipolar 1 disorder, schizophrenia, and schizoaffective disorder (128 cases and 201 controls), and the broad definition added bipolar 2 disorder and bipolar disorder NOS (217 cases and 201 controls). The mean age (standard deviation) at participation was 56.3 (16.2) years for cases and 60.0 (15.5) years for controls; most subjects had traversed much of the period of lifetime risk for these disorders. Genomic assays were done on N = 418 individuals, including SNP arrays (N = 418), WES (N = 26), WGS (N = 10), and karyotyping (N = 9). Of the 418 subjects, 300 individuals were part of 72 first-degree relative clusters. Evaluation of genome-wide SNPs confirmed expected relationships. As anticipated, many pedigree members had low levels of relatedness . People born in the northermost counties of Sweden had features consistent with a genetic isolate, and this was particularly pronounced in the Northern Swedish pedigree. Members of the pedigree had high levels of runs of homozygosity and genome-wide SNP homozygosity similar to or greater than that found in Finland , we performed clinical karyotyping of nine individuals from across the branches of the pedigree . All had normal karyotypes suggesting that a microscopic structural variant was unlikely to explain inheritance of major psychiatric disorders in this pedigree Figure .31. We identified three cases with three different structural variants  a ms Figure . We evalHLA-DRB5 (deletion and duplication), PRR21 (deletion), HLA-A (duplication), and SMC1B (duplication). No inversion breakpoints disrupted coding regions of any gene. Overall, we identified no structural variants suspected to account for the elevated rate of psychosis in this pedigree.Third, to ensure that we were not missing structural variants below detection thresholds for karyotyping and SNP arrays, we conducted whole-genome sequencing (30x coverage) of 10 cases  from across the pedigree. We manually inspected all CNVs called in ≥8 of 10 cases and not found in the 1000 Genomes Project (phase 3). This included 115 deletions (median size 1.1 kb), 38 duplications (median size 961 bp), and 72 inversions (median size 85.8 kb). Most of these structural variants overlapped segmental duplications or gene-poor regions of the genome and were likely false positives or inconsequential. The genes that had coding regions affected were C9orf72 and frontotemporal dementia) using ExpansionHunter along with a similar in-house approach (that previously identified a pathogenic Huntington’s disease CAG-repeat expansion33). None were identified.Using the PCR-free whole-genome sequencing data from 10 cases, we searched for known pathogenic trinucleotide repeat polymorphisms . These regions contained 2.58% of the SNPs used by pLDSC but were not enriched for the SNP-heritability of bipolar disorder  or major depression . Contrary to expectations, the SNP-heritability of schizophrenia was significantly depleted in these regions .The linkage regions  were unremarkable. Bioinformatic analysis using GREATP < 0.01). Bioinformatic analysis using GREAT34 showed no enrichments for any annotation. These regions were not enriched for SNP-heritability of bipolar disorder , schizophrenia , or major depression . Analysis of regions with case-control difference at P < 0.1 showed similar results. Statistical analysis of these regions using the broad and narrow definitions of illness showed no significant findings  either unknown or had a maximum allele frequency in any 1000 Genomes Project or GnomAD cohort <1e-5;36 (b) had predicted “high” impact or were located in a conserved coding region ( >ninetieth percentile);37 and (c) located in a brain-expressed gene (14 K genes defined as expression >1 in any GTEx brain sample38). This yielded 102 variants. Manual review of this list did not yield any high-confidence predictions that were present in more than half of the cases.We evaluated WES data from 26 cases from across the pedigree , and identified 129,651 variants (1.4% novel). We annotated variants passing quality control using the Variant Effect Predictor22. We contrasted three groups: cases from the pedigree , controls from the pedigree (N = 201), and an independent set of controls with no lifetime serious mental illness from Northern Sweden (N = 1267). The schizophrenia GRS data are depicted in Fig. F2,1593 = 9.06, P = 1.22e-4). Post-hoc mean testing using Tukey-Kramer HSD showed that pedigree cases had significantly greater schizophrenia GRS than pedigree controls  and population controls , but that pedigree controls were similar to population controls . Figure F2,1593 = 3.39, P = 0.034). Post-hoc tests showed that pedigree cases have slightly greater bipolar GRS than pedigree controls , but that the other two comparisons were not significantly different . These results provide a potential explanation for the clustering of major psychiatric illnesses within the Northern Swedish pedigree.We evaluated the contribution of common variation in the Northern Swedish pedigree using GRS. The training sets for schizophrenia and bipolar disorder were from the PGC after removing Swedish studiesDensely affected pedigrees can result from several genetic processes. The two extreme possibilities are that affected individuals inherit single variants of strong effect from common ancestors or that they inherit unusually high numbers of common variants of subtle effect. Our comprehensive analyses of many potential types of rare variation did not yield any candidate loci of strong effect . Given that our sample has features of a genetic isolate, we may have maximized our chances for detecting such variants.7 and from our prior study of a pedigree dense with bipolar disorder7. In this and in our prior study, the pedigrees were from geographically isolated areas with low migration rates. We speculate that founders had above average GRS and that subsequent matings with individuals from the same region served to increase GRS (on average). This process of “concentrating” GRS over generations could then increase the risk of affection in pedigree members. A compatible hypotheses was forwarded by de Jong et al. in a genetic study of a large pedigree dense with mood disorders39.Our results were consistent with inherited burden of common variation given that pedigree cases had somewhat higher schizophrenia and bipolar GRS compared to pedigree and community controls. We observed stronger prediction from schizophrenia GRS than from bipolar GRS, which we attribute to the greater informativeness of the schizophrenia GRS training set. The finding of higher GRS in pedigree cases was anticipated from theory40. This was leveraged in Scandinavia to identify RBM12 as a risk locus for psychosis41. Indeed, the PGC has scaled this approach: using its global networks, the PGC is in the process of ascertaining and genetically evaluating 100 densely affected pedigrees1. It is probable that most will result from concentrations of common variation but even one confident identification of a causal rare variant would be an important advance.The genetic study of dense pedigrees is a logical complementary strategy for gene-finding in psychiatric geneticsWe note that we cannot exclude the presence of risk variants with more complex inheritance patterns, variants with cryptic functional effects, or variants missed by our genomic assays. However, the presence of many common-variant risk alleles in a pedigree is a potential explanation for a dense pedigree.Supplemental Figures S1–S8


Underlying mechanisms of antiproliferative effects were found to be resulting from cell cycle regulation. SMT downregulated expression of cyclin D1-CDK4 and cyclin E-CDK2 complexes and upregulated p21waf1/cip1 and p27kip1. SMT also suppressed migration of SMCs against TNF-α stimulation. This is thought to have resulted from suppressing MMP2 and MMP9 expressions and ROS production. In summary, SMT attenuates abnormal migration of vascular smooth muscle cells via regulating cell cycle and suppressing MMPs expression and ROS production. Our study suggests that SMT, a traditionally used herbal formula, protects vascular smooth muscle cells and might be used as an antiatherosclerotic drug.Samul-Tang (SMT), consisting of four medicinal herbs, is a well-known herbal prescription treating hematological disorders related symptoms. Our previous study demonstrated that SMT attenuated inflammation of vascular endothelial cells. In condition of retained vascular dysfunction, vascular inflammation is initiated and results in activation of smooth muscle cells (SMCs). Activated SMCs lose control of cell cycle regulation and migrate into intima, resulting in formation of atheroma. Here, we further investigated whether SMT suppresses proliferation and migration of SMCs. SMT showed antiproliferative effects on SMCs by suppressing [ The vascular smooth muscle cells (SMCs) are located in the walls of blood vessels and regulate contraction or relaxation of blood vessel. Besides, SMCs are also involved in developing cardiovascular complication such as atherosclerosis resulting from chronic vascular inflammation or abnormal proliferation and migration of SMCs .waf1/cip1 and p27kip1 stabilize cyclin-CDK complexes and negatively regulate cell cycle -thymidine was added . After 24 h of incubation, cells were washed once with 2 ml of ice-cold PBS for 10 min, extracted three times with 2 ml of cold 10% TCA for 5 min each, and solubilized for at least 30 min at room temperature in 0.2 ml of 0.3 N NaOH, 1% SDS. After neutralization with 0.2 ml of 0.3 N HCl, [3H]-thymidine activity was measured in a liquid scintillation counter . Each experiment was conducted either in triplicate or in quadruplicate.Quiescent cells were treated with 10 ng/ml TNF-2O, blocked with 5% skimmed milk powder in Tris-Buffered Saline Tween-20 (TBS-T)  for 1 h, and incubated with the appropriate primary antibody at dilutions recommended by the supplier. Then the membrane was washed, and primary antibodies were detected with secondary antibodies conjugated to horseradish peroxidase, and the bands were visualized with enhanced chemiluminescence . Protein expression levels were determined by analyzing the signals captured on the nitrocellulose membranes using the ChemiDoc image analyzer .Cell homogenates were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Blots were then washed with Hα at 37°C for 24 h. After incubation, the microscopic photographs of migrated cells were measured by microscopy .The cell migration assay was evaluated by wound healing assay. Briefly, SMCs were plated in 12-well culture plates and cultured in DMEM containing 10% FBS. Scratches were performed with a sterile tip in a 12-well plate containing cells at 80% confluence level. Next, SMCs were pretreated with SMT for 30 min, which was followed by treatment with TNF-α for 24 h. The supernatant, conditioned medium was collected for zymography. SDS-PAGE for measurement of MMP2 activity was added with 0.1% gelatin in the 10% separated gel. The gel was washed with renaturation buffer (2.5% Triton X-100 in DW) at room temperature for 1 h and then incubated with development buffer  at 37°C overnight. Next, the gel was stained with 0.2% Coomassie brilliant blue R stain reagent at room temperature for 1 h. After washing with destain buffer, the gels were scanned using a ChemiDoc image analyzer .SMCs were pretreated with SMT for 30 min and stimulated with TNF-2DCFDA), was used to determine the intracellular generation of ROS by stimulation of TNF-α. Briefly, the confluent SMCs in the 6-well culture plates were pretreated with or without SMT for 30 min. After removing the SMT from the wells, the cells were incubated with 20 μM of CM-H2DCFDA for 1 h. The cells were stimulated with TNF-α, and the fluorescence intensity was measured at an excitation and emission wavelength of 485 nm and 530 nm, respectively, using a flow cytometry on FACSCalibur .The fluorescent probe, (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester) , Whereas SMT 10 μg/mL (#p < 0.05), 30 μg/mL (#p < 0.05), and 50 μg/mL (##p < 0.01), respectively, significantly suppressed incorporation of [3H]-thymidine against TNF-α stimulation in a dose-dependent manner.To investigate the effects of SMT on proliferation of TNF-α stimulation, signaling pathways of cyclin-CDK complexes and CDK inhibitors were assessed by western blot. As shown in α for 24 h significantly upregulated expression of cyclin D1 (∗∗p < 0.01) and CDK4 (∗p < 0.05) compared to untreated control group, Whereas SMT 30 μg/mL (#p < 0.05) and 50 μg/mL (##p < 0.01) significantly suppressed expression of cyclin D1 against TNF-α stimulation in a dose-dependent manner. Also, CDK4 expression was suppressed by SMT 50 μg/mL treatment (#p < 0.05) against TNF-α stimulation. As shown in α for 24 h significantly upregulated expression of cyclin E (∗∗p < 0.01) and CDK2 (∗∗p < 0.01) compared to untreated control group, whereas SMT 30 μg/mL (#p < 0.05) and 50 μg/mL (##p < 0.01) significantly suppressed expression of cyclin E against TNF-α stimulation in a dose-dependent manner. Also, CDK2 expression was suppressed by SMT 50 μg/mL treatment (#p < 0.05) against TNF-α stimulation. As shown in waf1/cip1 and p27kip1 (CDK inhibitors) were both significantly downregulated by stimulation with TNF-α for 24 h (∗∗p < 0.01), whereas SMT 50 μg/mL significantly inhibited downregulation of both p21waf1/cip1 and p27kip1 against TNF-α stimulation.To investigate mechanisms of antiproliferative effects of SMT on SMCs against TNF-These results demonstrate that SMT regulates cell cycle of SMCs via inhibiting cyclin and CDK expression and upregulating CDK inhibitors.μm (40x magnification). As shown in α stimulated SMCs showed increased migration compared to untreated control group, whereas SMT-treated SMCs (10–50 μg/mL) showed decreased migration against TNF-α stimulation.Migration of SMCs is closely related to its proliferation and is a previous process of neointima and plaque formation. Migration assay was performed under examination with microscope and scale bar indicates 500 α both increased compared to untreated control group, whereas SMT (10–50 μg/mL) suppressed secretion of MMP2 and MMP9 against TNF-α stimulation on SMCs.MMP2 and MMP9 are involved in the neointima formation. Gelatin zymography and western blot were performed to measure MMP2 and MMP9 levels. Zymography was performed with cell culture medium incubated with SMCs. As shown in α stimulation compared to untreated control group (∗p < 0.05), whereas SMT 50 μg/mL significantly suppressed protein expression of both MMP2 and MMP9 against TNF-α stimulation (#p < 0.05).As shown in 2DCFDA as described in Materials and methods. As shown in α stimulation (∗∗p < 0.01), whereas ROS scavenger N-acetyl-L-cysteine  inhibited production of intracellular ROS and SMT (10–50 μg/mL) also suppressed production of intracellular ROS (##p < 0.01), implicating its antioxidant effects.Oxidant stress is a major cause of endothelial dysfunction and vascular inflammation. To measure intracellular ROS production, SMCs were labeled with CM-Hα stimulation. We stimulated SMCs with TNF-α to make atherogenic circumstances. Cytokines such as TNF-α work as an autocrine and paracrine mediator and are highly expressed in atherosclerotic lesions [α also leads to production of other inflammatory factors inducing proliferation and migration of SMCs [3H]-thymidine incorporation assay in this study, which is used as an index of DNA synthesis. SMT significantly suppressed SMCs [3H]-thymidine incorporation against TNF-α stimulation in a dose-dependent manner. Thus, detailed antiproliferative mechanism of SMT on TNF-α stimulated SMCs cell cycle was investigated.This study demonstrates that SMT suppressed abnormal increase of SMCs proliferation and migration via regulating cell cycle, ROS production, and MMPs expression against TNF- lesions . Besides of SMCs . Prolife Streptomyces hygroscopicus [kip1 and inhibition of CDK activity [α stimulation. Furthermore, p21waf1/cip1 and p27kip1 (CDK inhibitors) expression of SMCs after TNF-α stimulation was significantly decreased. However, SMT also showed cell cycle regulative effects by significantly suppressing downregulation of p21waf1/cip1 and p27kip1 against TNF-α stimulation.Phases of the cell cycle are coordinated by CDKs which have little activity in the absence of cyclins. CDKs bind cyclins to form cyclin-CDK complexes and their activities are up to phosphorylation of CDKs and cyclin expression. In quiescent G0 phase, E2F family members exist in inactive form with retinoblastoma protein. However, after mitogenic stimulation, cyclin D1-CDK4 and cyclin E-CDK2 complexes phosphorylate retinoblastoma, dissociating E2F to express other cyclins and CDKs . Cell cyscopicus , is knowscopicus . Its proactivity . In thisα stimulation. In addition to cell cycle regulative effects of SMT, underlying mechanism of this phenomenon seems to be resulting from MMP suppressive effects of SMT. SMCs can produce proteolytic enzymes related to the remodeling of MMPs and ECM. In particular, MMP2 and MMP9 belong to representative MMPs. Recent knock-out study also suggests that MMP2 [α stimulation. In addition, SMT significantly suppressed the protein expression of both MMP2 and MMP9 against TNF-α stimulation.Besides, results of migration assay with microscopy showed that SMT suppressed SMCs migration against TNF-hat MMP2  and MMP9hat MMP2  are cruchat MMP2 . In this in vitro study suggests that oxidative stress may enhance MMP expression and activity [α stimulation increased the ROS production of SMCs and this might be influence the overall signaling pathways of proliferation and migration. In this regard, SMT suppressed production of intracellular ROS on TNF-α stimulated SMCs and it might contribute to cell cycle regulation and MMPs suppression by SMT.It is suggested that there is a certain relation between oxidative stress and MMPs modulation. Recent studies suggest that MMP-mediated ECM remodeling could be modulated by ROS and oneactivity . In thisα stimulation via inhibiting activation of nuclear factor-κB and inducing heme oxygenase-1 [Our previous study demonstrated that SMT attenuated inflammation of vascular endothelial cells against TNF-genase-1 . In condgenase-1 , 28. LikTo summarize, SMT attenuated abnormal migration of vascular smooth muscle cells via regulating cell cycle and suppressing MMPs expression and reactive oxygen species production. We suggest that SMT, a traditionally used herbal formula consisting of four herbs, might act as a vascular protective drug.