Amblyceps waikhomi sp. nov. is described from the Nongkon stream which drains into the Noa Dehing River, a tributary of the Brahmaputra River, in Arunachal Pradesh, India. The new species can be distinguished from congeners (except A. torrentis) in having a deeper body depth at anus. It further differs from congeners (except A. mangois and A. serratum) in having fewer vertebrae, from A. mangois in lacking (vs. having) strongly-developed projections on the proximal lepidotrichia of the median caudal-fin rays, and in having a longer, wider, and deeper head; and from A. serratum in having a posteriorly smooth (vs. with 4–5 serrations) pectoral spine, and unequal jaw length . It additionally differs from A. murraystuarti, A. torrentis, A. apangi, A. laticeps, and A. cerinum in having a deeply forked (vs. emarginate or truncate) caudal fin. This species is the seventh amblycipitid species known to occur in the Ganga-Brahmaputra River system. Amblyceps Blyth are small-bodied, elongate catfishes, occurring in fast flowing streams and rivers of mainland Southeast Asia and the Indian subcontinent [Fishes of the genus ontinent . The genontinent .Amblyceps are presently considered valid viz. A. apangi Nath and Dey, A. arunachalensis Nath and Dey, A. caecutiens Blyth, A. carinatum Ng, A. cerinum Ng and Wright, A. foratum Ng and Kottelat, A. kurzii (Day), A. laticeps (McClelland), A. macropterus Ng, A. mangois (Hamilton), A. murraystuarti Chaudhuri, A. platycephalus Ng and Kottelat, A. protentum Ng and Wright, A. serratum Ng and Kottelat, A. tenuispinis Blyth, A. torrentis Linthoingambi and Vishwanath, A. tuberculatum Linthoingambi and Vishwanath, and A. variegatum Ng and Kottelat [Eighteen species of Kottelat .Amblyceps were collected from a stream flowing into Noa Dehing River of the upper Brahmaputra basin in Namsai District. After a detailed comparison with all 18 congeners, we concluded that they represent an unnamed Amblyceps species, which is herein described as Amblyceps waikhomi sp. nov.During recent ichthyological surveys of the eastern part of Arunachal Pradesh, five specimens of Measurements were made on the left side of specimens with a digital caliper to the nearest 0.1 mm and fin rays were counted under a Nikon SMZ800 stereo-zoom microscope. Morphometric measurement and fin ray counts followed Ng and Wright . Measure2), alkalinity, and hardness followed APHA [The water quality of the habitat was analysed by taking three replicates randomly from the type locality of the new species during January 2014. Estimation of dissolved carbon dioxide  can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "Amblyceps waikhomi sp. nov.urn:lsid:zoobank.org:act:8F9219B2-619E-4321-8C2E-F31926DAD0FFHolotype. ZSI/APRC/P-1125, 42.9 mm SL; India, Arunachal Pradesh, Namsai District, Nongkon stream at Nongkon village draining into Noa Dehing River (Brahmaputra basin), 27°36’05”N, 95°50’51”E; Akash Kachari, 5 October 2013.Paratypes. Locality and collector as for holotype, RGUMF 269, 40.2 mm SL, 1 cleared and stained (c&s), 20 June 2014; RGUMF 270, 37.4 mm SL, 1 c&s, 12 October 2014; RGUMF 271, 30.4–44.7 mm SL, 2, 6 December 2014.Amblyceps waikhomi sp. nov.(A. mangois and A. serratum) total vertebrae (34–35 vs. 37–48) (A. mangois in lacking (vs. having) strongly-developed projections on the proximal lepidotrichia of the median caudal-fin rays and in having a longer, wider, and deeper head (A. serratum in having a posteriorly smooth (vs. with 4–5 serrations) pectoral spine, and unequal jaw length . It additionally differs from A. murraystuarti, A. torrentis, A. apangi, A. laticeps, and A. cerinum in having a deeply forked (vs. emarginate or truncate) caudal fin. sp. nov., S1 Fig . 37–48) . It diffper head   gill rakers. Gill membranes narrowly joined at isthmus, with 10 (n = 2) branchiostegal rays.Dorsal fin with a spinelet, a spine, and 6 (n = 5) branched rays; its origin closer to snout tip than to adipose-fin origin; posterior margin of fin convex; fin base fleshy and swollen. Dorsal-fin spine smooth, short and straight, distal tip sharply pointed, its length reaching one-third of fin height. Adipose fin short, low, commencing from vertical midway between anus and anal-fin origin, posterior margin well separated from caudal fin. Pectoral fin with a smooth spine and 6 (n = 5) branched rays; origin anterior to vertical through posterior margin of operculum posterior margin convex. Pectoral-fin spine longer than dorsal-fin spine, straight, anterior and posterior margins smooth. Skin covering pectoral-fin base and skin covering spine swollen. Pelvic fin with i–ii,4–5 rays, tip of adpressed fin reaching beyond urogenital opening but not anal-fin origin. Anal fin with iii,10 rays. Caudal fin deeply forked with i,7,8,i (n = 5) principal rays, simple-principal and segmented procurrent rays of upper and lower lobe bears pinnate like rays anteriorly, upper lobe longer than lower .In 70% ethanol: Dorsal and lateral surfaces of head and body brownish, ventrally creamy.Presently known only from its type locality, Nongkon stream draining to Noa Dehing River (Brahmaputra basin), Namsai District, Arunachal Pradesh .2 1.53 mg/l, alkalinity 66.06 mg/l, and hardness 71.8 ± 3.05 mg/l; while the physical parameters were pH 6.78, air temperature 23.8 ±0.87°C, water temperature 23.5 ±0.96°C, transparency 79.5 ±1.93 cm,and conductivity 173.33 μS.The new species was collected from a slow moving stream (water current 0.16 m/s) with a bottom substrate dominated by sand, occasionally associated with mud . The speThe new species is named after Waikhom Vishwanath, honouring his outstanding contribution to freshwater ichthyology in the Indian subcontinent.Amblyceps species have unossified pinnate-like rays (4–5 pinnate rays per lepidotrichium) on the anterior margins of the procurrent caudal-fin rays [A. mangois, A. arunachalensis, A. apangi, and A. waikhomi and found the pinnate like rays (except A. apangi) only on the distal half of the anterior margin of the segmented procurrent rays and unbranched principal rays. Ng and Kottelat [Amblyceps of the Indian subcontinent. This finding was confirmed by our observations on A. mangois , A. waikhomi further differs from A. tenuispinis in having a shorter snout (23.4–27.2% HL vs. 33.6–43.3) and dorsal to adipose distance (17.0–21.3% SL vs. 23.9–34.0), a longer pectoral fin (16.8–19.0% SL vs. 15.1–16.6), and a deeper caudal peduncle (13.0–16.4% SL vs. 9.6–12.9); from A. arunachalensis in having a longer predorsal (25.7–30.6% SL vs. 22.5–23.2), prepelvic (48.3–50.3% SL vs. 45.1–45.9), prepectoral (19.2–23.0% SL vs. 18.2–19.8), and adipose-fin base (20.3–23.7% SL vs. 18.1–19.8) lengths and fewer pleural ribs (7 vs. 12); and from A. macropterus in having fewer vertebrae (34–35 vs. 37), a shorter adipose-fin base (20.3–23.7% SL vs. 28.3), and in lacking (vs. having) the strongly-developed projections on the proximal lepidotrichia of the median caudal-fin rays.With the description of Amblyceps waikhomi can be further distinguished from A. carinatum by its shorter adipose-fin base length (20.3–23.7% SL vs. 37.5–44.6) and longer dorsal to adipose distance (17.0–21.3% SL vs. 7.8–10.7); from A. tuberculatum by its shorter caudal peduncle length (15.5–18.6% SL vs. 21.2–22.4), shorter dorsal to adipose distance (17.0–21.3% SL vs. 27.8–28.0), and incomplete (vs. complete) lateral line; from A. kurzii by its longer adipose-fin base (20.3–23.7% SL vs. 15.1–18.3), shorter dorsal to adipose distance (17.0–21.3% SL vs. 30.1–30.6), and deeper caudal peduncle (13.0–16.4% SL vs. 9.8–10.7).Amblyceps waikhomi can be further distinguished from A. platycephalus by its fewer principal caudal-fin rays (17 vs. 20); from A. caecutiens in having a larger eye (6.7–7.5% HL vs. 2.0–3.4) and shorter adipose-fin base (20.3–23.7% SL vs. 25.6–33.5); from A. protentum in having a longer prepelvic (48.3–50.3% SL vs. 42.8–47.8), prepectoral (19.2–23.0% SL vs. 15.9–18.3), and pectoral-fin (16.8–19.0% SL vs. 11.2–14.4) lengths, shorter and deeper caudal peduncle , shorter snout (23.4–27.2% HL vs. 30.1–34.6), and shorter dorsal to adipose distance (17.0–21.3% SL vs. 26.3–32.2); and from A. variegatum by its uniformly brownish (vs. mottled) body coloration.Amblyceps mangois: ZSI-NRS/F2556, 31,40.0–49.4 mm SL, India: Uttar Pradesh, Saharanpur, Padhoe River at Kalsia Ghat, Ganga River basin, K.P. Singh, 20 January 1972. RGUMF, unregistered, 3, 45.2–55.5 mm SL, India: Uttarakhand, Nainital district, Gola River at Kathgodam, Ganga River basin, A. Darshan, 30 April 2011. MUMF 14301–14302, 2, 35.5–37.1 mm SL, India: Barak River at Silchar (Assam), M. Shantakumar and K. Nebeshwar, 16 December 2000. MUMF 14061, 1, 48 mm SL, India: Arunachal Pradesh, Dikrong River, K. Nebeshwar.Amblyceps arunachalensis: RGUMF 117, 82.6–97.2 mm SL, 3, India: Arunachal Pradesh, Subansiri River at Daporijo, Brahmaputra basin, 7 June 2005.Amblyceps apangi Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigLubpr: lowermost upper branched principal ray; Sdp: strongly developed-projections (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Continuous deep sedation at the end of life is a practice that has been the topic of considerable ethical debate, for example surrounding its perceived similarity or dissimilarity with physician-assisted dying. The practice is generally considered to be legal as a form of symptom control, although this is mostly only assumed. France has passed an amendment to the Public Health Act that would grant certain terminally ill patients an explicit right to continuous deep sedation until they pass away. Such a framework would be unique in the world.In this paper we will highlight and reflect on four relevant aspects and shortcomings of the proposed bill. First, that the bill suggests that continuous deeps sedation should be considered as a sui generis practice. Second, that it requires that sedation should always be accompanied by the withholding of all artificial nutrition and hydration. In the most recently amended version of the legal proposal it is stated that life sustaining treatments are withheld unless the patient objects. Third, that the French bill would not require that the suffering for which continuous deep sedation is initiated is unbearable. Fourth, the question as to whether the proposal should be considered as a way to avoid having to decriminalise euthanasia and/or PAS or, on the contrary, as a veiled way to decriminalise these practices.The French proposal to amend the Public Health Act to include a right to continuous deep sedation for some patients is a unique opportunity to clarify the legality of continuous deep sedation as an end-of-life practice. Moreover, it would recognize that the practice of continuous deep sedation raises ethical and legal issues that are different from those raised by symptom control on the one hand and assisted dying on the other hand. Nevertheless, there are still various issues of significant ethical concern in the French legislative proposal. Continuous deep sedation at the end of life (CDS) is the practice whereby a physician uses sedative medication resulting in the reduction or elimination of a patient’s consciousness until she dies. This practice is the topic of considerable ethical debate –3. Part Currently the debate on sedation is very lively in France, where an amendment to the Public Health Act was made to include an explicit right for terminally ill patients to 'deep and continuous sedation' . This hasui generis practice. Second, that it requires that sedation should normally be accompanied by the withholding of all life sustaining treatment. Whether this also includes artificial nutrition and hydration is, as will be discussed below, a matter of considerable debate. Third, that the French bill would not require that the suffering for which continuous deep sedation is initiated is severe or unbearable. Fourth, the question as to whether the proposal should be considered as a way to avoid having to decriminalise euthanasia and/or PAS or, on the contrary, as a veiled way to decriminalise these practices.This paper will elaborate on the current ethical and legal debate in France and on the unique and ground-breaking nature of the legislative proposal on continuous sedation. We will highlight and reflect on four ethically relevant aspects and shortcomings of the bill. First, that the bill suggests that continuous deep sedation should be considered as a Loi Leonetti (Leonetti Law) after the man who originally proposed the law— which amends the Public Health Act . This law explicitly allows physicians to provide far-reaching symptom control, even at the risk of shortening life, while prohibiting physician-assisted suicide and euthanasia. It has been suggested that the Loi Leonetti also allows continuous deep sedation as a form of far-reaching symptom control ur anxiety that [sedation] may be confused with euthanasia encourages us to obscure or sanitize the features both practices share .Debating continuous deep sedation together with medically-assisted dying involves considerable risks. If enacting an explicit right to this practice is indeed a disguised way to decriminalise euthanasia or physician-assisted dying, then it is intellectually dishonest. If, instead, the aim of creating a right to continuous deep sedation is to avoid decriminalising medically-assisted dying, this poses risks of its own. As noted by Battin (2008):sui generis practice could remove the need to portray the practice either as symptom control or as a form of euthanasia. This might make it less difficult to focus on the issues that are most relevant and pertinent to continuous deep sedation.France is currently in the midst of a fierce debate on what constitutes a dignified end of life and which practices are acceptable for physicians to perform. At the very core of the debate is the practice of continuous deep sedation. As argued in this paper, what is playing out is a unique opportunity to clarify the legality of continuous deep sedation as an end-of-life practice. Recognizing continuous deep sedation as a However, there are still various issues of significant ethical concern in the French legislative proposal. Automatically withholding artificial nutrition and hydration is in our view overly strict and unjustifiably equates artificial nutrition and hydration with medical treatment. As argued above, there is also danger in seeing it as care that can never be withdrawn. Focussing only on the question as to whether ANH should be seen as just another treatment or as fundamental basic care seems to ignore the most crucial question, i.e., in what circumstances it may justifiably be withdrawn or withheld.Second, by not limiting the right to continuous deep sedation to patients with severe or unbearable suffering, the French proposal leaves open the possibility to request continuous deep sedation for mild and bearable suffering. In our view, continuous deep sedation is a far reaching practice that can only be justified by reference to an ethical principle of proportionality. However, this ethical justification can only apply to those cases where suffering is both refractory and very severe or unbearable.sui generis practice and attempts to portray the practice as either similar or dissimilar from medically assisted dying risks ignoring patients’ actual interests in having a peaceful and dignified end of life.Finally, the debates in France demonstrate that discussing continuous deep sedation often also raises the issues of physician-assisted suicide and euthanasia. Opponents of physician-assisted suicide might see continuous deep sedation as a first step on the slippery slope. Others object to the focus on continuous sedation as an excuse to ignore the most pressing issues at the end of life. Continuous deep sedation then proves to be an unacceptable compromise to both proponents and opponents of euthanasia and/or physician-assisted suicide. Moreover, a failure to recognize sedation as a We believe France’s attempt to reflect on continuous deep sedation as a practice that needs clarification provides an interesting example. Most importantly, continuous deep sedation should not be seen as a catch all solution to ethical issues at the end of life, for it is not the automatically preferable alternative it is sometimes portrayed to be . As ment


Background. Intrauterine environment plays a pivotal role in the origin of fatal diseases such as diabetes. Diabetes and obesity are associated with low-grade inflammatory state and dysregulated adipokines production. This study aims to investigate the effect of maternal obesity and malnutrition on adipokines production  in F1 offspring in rats. Materials and Methods. Wistar rats were allocated in groups: F1 offspring of control mothers under control diet (CF1-CD) and under high-fat diet (CF1-HCD), F1 offspring of obese mothers under CD (OF1-CD) and under HCD (OF1-HCD), and F1 offspring of malnourished mothers under CD (MF1-CD) and under HCD (MF1-HCD). Every 5 weeks postnatally, blood samples were obtained for biochemical analysis. Results. At the end of the 30-week follow-up, OF1-HCD and MF1-HCD exhibited hyperinsulinemia, moderate dyslipidemia, insulin resistance, and impaired glucose homeostasis compared to CF1-CD and CF1-HCD. OF1-HCD and MF1-HCD demonstrated low serum levels of adiponectin and high levels of leptin compared to CF1-CD and CF1-HCD. OF1-CD, OF1-HCD, and MF1-HCD had elevated serum levels of TNF-α compared to CF1-CD and CF1-HCD (p < 0.05). Conclusion. Maternal nutritional manipulation predisposes the offspring to development of insulin resistance in their adult life, probably via instigating dysregulated adipokines production. Increasing evidence from clinical and epidemiological research suggests that prenatal environment plays a crucial role in the origin of fatal diseases such as the metabolic syndrome and its components: insulin resistance, hypertension, and dyslipidemia . MaternaAdiponectin is an insulin-sensitizing, antidiabetic, antioxidant, anti-inflammatory, and antiatherogenic adipokine, which acts on adiponectin receptors, for example, AdipoR1 and AdipoR2 to exert its actions . Leptin α (TNF-α) is an inflammatory cytokine that has a variety of functions such as mediating apoptosis and regulation of immune system. TNF-α exerts its effects via its action on TNFR1 and TNFR2 receptors [α level is elevated in obese and diabetic patients and is correlated with various complications of diabetes [Subclinical inflammation is a hallmark of obesity, diabetes, and metabolic syndrome . Tumor neceptors . Serum Tdiabetes , 8.α) in F1 offspring in rats, as a potential mechanism for the intergenerational effect of maternal nutritional environment on the offspring.Epigenetic modifications, deterioration of glucose tolerance, elevated inflammatory markers, and excessive oxidative stress could be possible mechanisms for the intrauterine programming of fetal diseases , 9, 10; The study was done in accordance with the ethical guidelines of the Medical Research Institute, Alexandria University, Alexandria, Egypt. Wistar rats were housed as 4 per cage at an ambient temperature of 23 ± 1°C with 12/12 h light/dark cycles and 45 ± 5% humidity.Female rats were allocated randomly in three groups: control, obese, and malnourished. Obesity was instigated via maintaining female neonates under obesogenic diet for two months after weaning. Female rats that were 20% heavier than controls of the same age were considered obese. Malnutrition was instigated through maintaining female neonates under low-protein diet (8% protein) for two months after weaning. Female rats that were 20% lighter than controls of the same age were considered malnourished.Pregnancy was carried out by overnight mating the females  with normal healthy males. Pregnancy was confirmed next morning by the presence of vaginal mucus plug. Pregnancies were completed to term. After delivery, the offspring were weaned to control diet (CD) or high-caloric diet (HCD) and followed up for 30 weeks. Therefore, the offspring groups were as follows: F1 offspring of control mothers under CD (CF1-CD), F1 offspring of control mothers under HCD (CF1-HCD), F1 offspring of obese mothers under CD (OF1-CD), F1 offspring of obese mothers under HCD (OF1-HCD), F1 offspring of malnourished mothers under CD (MF1-CD), and F1 offspring of malnourished mothers under HCD (MF1-HCD). Pregnancy outcome and composition of diets used were previously published . Every 5Fasting blood glucose (FBG) level was measured using glucometer . A commercial diagnostic kit (Randox (UK)) was used to assess lipid profile according to the manufacturer's instructions.α were assessed using ELISA kits  according to the manufacturer's instructions.Plasma insulin was assessed using ELISA kit (Mercodia). Serum levels of adiponectin, leptin, NEFA, and TNF- t-test was employed to compare the mean values of females and those of males of the same group at the same age. The results are presented as mean ± SD. Values of p > 0.05 were considered nonsignificantly different, while those of p < 0.05 were considered significant.All statistical analyses were performed using SPSS statistical software version 18 . The data were analyzed using the one-way analysis of variance (ANOVA) followed by LSD test to compare the mean values from the offspring of obese and malnourished mothers and the offspring of control mothers.At week 5, only males of OF1-HCD showed significantly higher FBG level than CF1-CD and CF1-HCD groups. At week 30, males and females of OF1-HCD and MF1-HCD exhibited significantly higher FBG levels than CF1-CD and CF1-HCD groups. Furthermore, males of OF1-CD had significantly higher FBG level compared to CF1-CD and CF1-HCD groups at the 30th week .At week 5, only females of OF1-HCD showed significantly higher insulin levels than CF1-CD and CF1-HCD groups. Also, females of MF1-CD and MF1-HCD groups showed higher insulin levels than their male counterparts . At weekμIU/mL) and glucose levels (mmol/L) indicated that males and females of OF1-HCD group as well as females of MF1-HCD were insulin resistant compared to CF1-CD and CF1-HCD groups at the 5th week  using fasting serum levels of insulin  level compared to CF1-CD. At week 15, males of OF1-HCD showed significantly higher TGs level compared to CF1-CD and CF1-HCD. Also, males of MF1-HCD exhibited significantly higher TGs level compared to CF1-CD. At the 30th week, all members of CF1-HCD and OF1-HCD as well as males of OF1-CD, MF1-CD, and MF1-HCD exhibited elevated TGs level compared to CF1-CD. Moreover, males of OF1-HCD showed elevated TGs level compared to CF1-HCD .Members of OF1-HCD showed significantly higher hepatic TGs content compared to CF1-CD from the 10th week to the 30th week, except males at the 10th week. Also, OF1-HCD showed significantly higher hepatic TGs content compared to CF1-HCD from the 10th week to the 30th week, except males at weeks 10 and 15. Members of OF1-CD as well as females of MF1-HCD exhibited significant rise in TGs content in the liver compared to CF1-CD at only the 30th week .Members of OF1-CD showed significantly higher muscular TGs content compared to CF1-CD at weeks 25 and 30. Also, females of this group had higher muscular TGs content compared to CF1-HCD at the last two time points. OF1-HCD group  showed significantly higher muscular TGs content compared to CF1-CD throughout the follow-up. Moreover, this group had significant rise in their muscular TGs content compared to CF1-HCD from weeks 10 and 30, except females at the 10th week. Members of MF1-CD and MF1-HCD demonstrated significantly elevated muscular TGs content compared to CF1-CD at the 30th week .At week 15, members of OF1-HCD and males of MF1-HCD showed significantly elevated total serum cholesterol level compared to CF1-CD. At week 30, members of CF1-HCD, OF1-HCD, and MF1-HCD as well as females of OF1-CD exhibited significantly elevated total serum cholesterol level compared to CF1-CD .At week 15, only females of OF1-HCD showed significantly lower serum high-density lipoprotein-cholesterol (HDL-C) levels than CF1-CD group. All members of CF1-HCD, OF1-HCD, and MF1-HCD as well as males of MF1-CD exhibited significantly lower serum HDL-C levels than CF1-CD group at the end of the follow-up period. Moreover, males of MF1-HCD demonstrated significant drop of their HDL-C levels compared to their female counterparts and CF1-HCD group .Regarding levels of low-density lipoprotein-cholesterol (LDL-C) levels, only males of OF1-HCD and MF1-HCD had significantly higher LDL-C levels than CF1-CD at the 15th week. Also, males of OF1-HCD showed significantly higher LDL-C levels than CF1-HCD at the 15th week .All members of CF1-HCD group had a significant rise in their serum nonesterified fatty acids (NEFA) levels from week 10 to week 30 compared to CF1-CD, except females at weeks 15, 25, and 30 that did not show such significant difference. Also, males of OF1-CD showed significantly higher NEFA levels at weeks 10, 20, 25, and 30 compared to CF1-CD. All members of OF1-HCD  showed higher NEFA levels compared to CF1-CD. Moreover, males of MF1-HCD from week 10 to week 30 and females of MF1-HCD at the 30th week demonstrated higher NEFA levels compared to CF1-CD .Females had significantly higher serum adiponectin levels than their male counterparts in all study groups throughout the study period. Males of CF1-HCD and OF1-CD showed significantly lower serum adiponectin levels than CF1-CD from week 20 to week 30. All members of OF1-HCD  showed significantly lower serum adiponectin levels than CF1-CD throughout the study period. Also, members of OF1-HCD had a significant drop in their serum adiponectin levels compared to CF1-HCD at weeks 25 and 30. Members of MF1-HCD  had significantly lower adiponectin levels compared to CF1-CD. Furthermore, females of MF1-HCD (at weeks 25 and 30) showed a significant drop in their adiponectin levels compared to CF1-HCD .Females had significantly elevated serum leptin levels than their male counterparts in all the study groups throughout the study period. Throughout the study, males of CF1-HCD (except at the 5th week), members of OF1-CD , and all members of MF1-HCD  had significantly higher leptin levels compared to CF1-CD. Moreover, females of OF1-CD (from week 10 to week 30), males of OF1-CD (at weeks 15 and 30), females of OF1-HCD , males of OF1-HCD (at weeks 15 and 20), and females of MF1-HCD (from week 10 to week 30) demonstrated higher leptin levels compared to CF1-CD .α levels than their male counterparts in all the study groups (except CF1-HCD and OF1-CD at the 5th week) throughout the study period. All members of CF1-HCD , all members of OF1-CD , all members of OF1-HCD, and all members of MF1-HCD  showed significantly higher serum TNF-α levels compared to CF1-CD throughout the study period. Moreover, members of MF1-CD demonstrated significantly higher serum TNF-α levels compared to CF1-CD from week 20 to week 30. All members of OF1-CD from week 10 to week 30  showed significantly higher serum TNF-α levels compared to CF1-HCD. Furthermore, all members of OF1-HCD  and MF1-HCD showed significantly higher serum TNF-α levels compared to CF1-HCD throughout the follow-up period  might interfere with insulin action by suppressing insulin signal transduction pathways [There is a strong association between markers of inflammatory activity and endothelial dysfunction, the trigger for cardiovascular diseases . Interespathways .In conclusion, maternal nutritional manipulation may predispose the offspring to development of insulin resistance in their adult life, probably via provoking a state of dysregulated adipocytokines production.


Drastic improvements in transmission rate and system capacity are required towards 5th generation mobile communications (5G). One promising approach, utilizing the millimeter wave band for its rich spectrum resources, suffers area coverage shortfalls due to its large propagation loss. Fortunately, massive multiple-input multiple-output (MIMO) can offset this shortfall as well as offer high order spatial multiplexing gain. Multiuser MIMO is also effective in further enhancing system capacity by multiplexing spatially de-correlated users. However, the transmission performance of multiuser MIMO is strongly degraded by channel time variation, which causes inter-user interference since null steering must be performed at the transmitter. This paper first addresses the effectiveness of multiuser massive MIMO transmission that exploits the first eigenmode for each user. In Line-of-Sight (LoS) dominant channel environments, the first eigenmode is chiefly formed by the LoS component, which is highly correlated with user movement. Therefore, the first eigenmode provided by a large antenna array can improve the robustness against the channel time variation. In addition, we propose a simplified beamforming scheme based on high efficient channel state information (CSI) estimation that extracts the LoS component. We also show that this approximate beamforming can achieve throughput performance comparable to that of the rigorous first eigenmode transmission. Our proposed multiuser massive MIMO scheme can open the door for practical millimeter wave communication with enhanced system capacity. The rapid diffusion of smartphones has triggered extensive diversification in mobile services. Not only is data traffic exploding, but also terminals are crowding public sites such as stations, airports and event venues. Unfortunately, existing frequency resources are depleted, especially in the microwave band, since many kinds of wireless communication systems such as wireless fidelity (Wi-Fi), worldwide interoperability for microwave access (WiMAX) or long-term evolution (LTE) (-Advanced) have turned into voracious consumers. Overcoming this shortfall is a critical issue in wireless communication. Drastic improvements of transmission rate and system capacity are required towards 5th generation mobile communications (5G) . PromisiChannel environments in the millimeter wave band are considered to be dominated by the Line-of-Sight (LoS) component since the base station (BS) or UEs are required to have highly directive antennas to obtain sufficient transmission/reception gain. In this situation, multiuser diversity gain is expected to increase the system capacity  since thHowever, critical issues remain with massive MIMO in the millimeter wave band; CSI per antenna between transmitter and receiver is noisy due to the short link budget as mentioned above. Moreover, the computation complexity imposed by eigenmode transmission is excessive due to singular value decomposition (SVD) calculation. This becomes especially serious in wideband systems. To realize 1st eigenmode reception at a UE with multiple antenna elements, the beam is simply steered toward the BS antenna array. To estimate angle of arrival (AoA) is the straightforward way and multiple signal classification (MUSIC)  or estimThis paper proposes an approximate beamforming scheme based on highly efficient CSI estimation. It is based on frequency domain CSI interpolation schemes using sparsely arranged training subcarriers, on which power density is concentrated. Although numerous interpolation schemes have been investigated ,21, our T, and (.)H represent absolute values, Frobenius norm, transpose and conjugate transpose, respectively.The rest of this paper is organized as follows. Nt element uniform planar array (UPA) serves Nu UEs with Nr element UPA where each UE is assigned Ns signal streams. To ensure the LoS environment and reduce the probability of human blockage  is the AoA or angle of departure (AoD) from the x-axis toward the positive z-axis, and θ [rad] is the angle from the positive y-axis toward the xz plane. pA [dB] is radiation pattern of the p-th antenna element. Assuming that from 3GPP 3D channel model . Note that Ui is obtained from the CSI at t = 0. UE speed is assumed to be 10 km/h and Rician K factor is 10 dB. Nr = 16. As shown, the correlations of the 2nd–4th eigenmodes rapidly decrease and fluctuate at a lower value while that of the 1st eigenmode is almost always 1. Multistream transmission per UE is considered to be severely affected by the channel time variation, especially in the 2nd and higher order eigenmodes. Nr, as the parameter. Increasing Nr can suppress the channel fluctuation impact. These fundamental results yield the expectation that 1st eigenmode transmission with large antenna arrays can achieve quite stable multiuser SDM performance.BS calculates the precoding weight via BD . Here, tAs discussed in the previous section, the 1st eigenmode is mostly formed by the LoS component (NLoS components are suppressed). Two key challenges are reviewed here; CSI estimation in the short link budget situation and computation complexity for SVD, which is required for each subcarrier. This section presents a simplified beamforming scheme by approximately extracting CSI from the LoS component in order to alleviate the calculation cost as well as the additive noise effect.10(2048/4/16) = 15.1 dB before beamforming. In addition, reducing the number of subcarriers can suppress PAPR per antenna element, which alleviates the need for input backoff (IBO) for training signal transmission. Second, each UE antenna element receives the training signals as if they had been transmitted from a single antenna element. UE then obtains CSI for received subcarriers using known training sequence and then calculates relative CSI, which represents the relationship of the CSI between the reference antenna element and the CSI of the other ones. Next, the phase component of the relative CSI is extracted. Third, phase information for null subcarriers is interpolated by linear regression. Let c, λ, cf (=c/λ), sf and md be the light speed, wavelength, carrier frequency, subcarrier spacing, and distance between the m-th UE antenna element and the BS, respectively; relative phase component of LoS channel at the k-th subcarrier, exp{mjφ(k)}, is expressed as,Δd = md − d1. Here, phase fluctuation in the frequency domain is due to the term exp{−j2π(skf/c)Δd}. In millimeter wave communication, inter-element space becomes much smaller than BS-UE distance so the relative phase information can be expressed as an array factor. Assuming a linear array with half wavelength spacing, Δd = (λ/2)(m − 1)sinθ where θ is AoA. From this we can derive:B is the channel bandwidth. It is obvious that −1/2 ≤ (skf/B) ≤ 1/2. When the system parameters are set to cf = 20 GHz and B = 400 MHz, fraction bandwidth, B/cf, is 0.02. We consider that m = 16 and θ = 45° at the limit, so in-band phase fluctuation is around 10% of 360°. Since this condition can be satisfied, linear regression can precisely extract the LoS component using extremely reduced training subcarriers. Relationship between mϕ(k) and k can be described as:ma and mb can be obtained by the well-known least squares method (LSM) as follows:k’ represents the subcarrier indices used for the training signal. Above operation is performed through the m-th element. Fourth, reception weight vector for the i-th UE is obtained as Ui = ϕi = T. Assuming time division duplex (TDD), UE should calibrate CSI for transmission beamforming since uplink and downlink signals go through different circuits, e.g., HPA and low noise amplifier (LNA). CSI for transmission can be obtained by applying a reciprocity calibration coefficient [H’, as shown in Equation (14). If necessary, the highly efficient CSI estimation shown in The proposed scheme is illustrated in fficient ,30. LastNc and Np indicate the subcarrier number for signal transmission and for training signal transmission in the proposed scheme, respectively. That of SVD is based on QR-decomposition and Householder reflections [lections .K = 10 dB, the multipath component is modeled as 11 path exponential decay with 3 dB attenuation for each 10 ns as per [Rtxi,, Rrxi,, and LoS channel, change with UE rotation on the horizontal plane. CSI is updated every 1.3 ms. This corresponds to 200 symbols, each with 6.67 μs symbol duration. CSI estimation error due to the receiver noise is excluded in order to evaluate the impact of the outdated CSI. We compare the following three schemes:Case 1: Four stream per UE via BD;Case 2: One stream per UE via BD (1st Eigenmode); andCase 3: One stream per UE via simplified beamforming .Principal simulation parameters are listed in s as per . SpatialIn Case 1, UE is assumed to perform minimum mean square error (MMSE) detection at every symbol reception. Cases 2 and 3 simply steer the beam to the BS array by using CSI at the update instance. We evaluate achievable signal-to-interference plus noise power ratio (SINR) and throughput performance. Throughput is determined based on modulation coding scheme (MCS) matches as the received SINR ,34. The NuNs = 16; 4 UEs with four streams for Case 1 and 16 UEs with single stream for Case 2. Maximum beamforming gain is calculated as 10log10{NtNr/(NuNs)} = 10log10(256 × 16/16) = 24 dB and the 1st eigenmode properly yields these gain values in Cases 1 and 2. However, higher eigenmodes for Case 1 exhibit lower SINR than the 1st one and they rapidly decrease as time progresses. Their achievable throughput values are lower than 1 bps/Hz at t = 1.3 ms. As Case 2 allocates a single stream per UE, SINR values of all signal streams remain high so the throughput degradation is minimal.First we observe time variant characteristics within the CSI estimation period. Note the achievable throughput values are directly converted from SINR in the evaluation. Practical MCS need to be determined with some margin considering the unpredictable SINR degradation possible with the time varying channel. If appropriate MCS cannot be selected, expected throughput cannot be achievable. The 1st eigenmode approach can suppress such MCS selection error since it realizes stable SINR performance. Therefore, the superiority of the 1st eigenmode transmission of Case 2 is expected to be demonstrated more clearly in actual use.The following examines the proposed beamforming scheme in Case 3. v. With Case 1, system throughput is decreased as movement speed increases and is reduced by 46.9% at 60 km/h. The reductions observed in Cases 2 and 3 are 6.8% and 10.4%, respectively. The gap between them is only 3.6%. Multiuser MIMO with 1st eigenmode transmission as well as our proposal can handle such high mobility situations, the movement speed is expected to be around 10 km/h considering the small cell use cases. It should be noted that extending the CSI feedback period M times corresponds to increasing the UE speed M times. Therefore, this evaluation is useful in understanding the impact of long CSI feedback periods from the viewpoint of control overhead savings.Nu, is evaluated and the result is shown in Nu = 16. In addition, Case 1 provides higher system throughput than Cases 2 and 3 at Nu < 12. This is because the total number of signal streams for Case 1 is four times those of Cases 2 and 3 under the same spatially multiplexed UE number. However, it devours the DoF resources and results in inefficient UE throughput. The system throughput finally decreases as Nu increases further. Meanwhile, the system throughput of Cases 2 and 3 linearly increases with Nu in the plotted region and they outperform Case 1 when Nu > 14. There remain enough DoF resources with single stream transmission per UE and all UEs exploit the 1st eigenmode, which provides the highest gain. Since UEs are spatially de-correlated, 1st eigenmodes can keep their high gain and thus higher spectral efficiency is achieved even in large number of Nu.Finally, average system throughput in terms of multiplexed UE number, L,NTBeam, NRBeam, and  be the number of scatters, transmission/reception training beams, and discrete angle sets, respectively, its complexity is estimated to be O(LNTBeamNRBeam(G02 + G12)), i.e., 37,365,760 if we set L = 10, NTBeam = NRBeam = 32,  =  as the derived parameters. The proposed scheme offers a 45.2% reduction in complexity. However, it should be understood that our proposal can ease hardware implementation since it does only require multiplications with matrices and a vector with simplified algorithms. This is one of the remarkable advantages of our proposal.Above results prove that exploiting the 1st eigenmode can realize stable multiuser MIMO transmission in time varying channels. System throughput can be significantly enhanced by spatially multiplexing a large number of UEs. Furthermore, we have confirmed the effectiveness of the proposed beamforming scheme based on simplified and high efficient CSI estimation. From the complexity estimate in 10(2048/4/16) = 15.1 dB. In addition, thinning out the training subcarriers also reduces PAPR and thus the transmission power can be raised thanks to easing IBO; this reduction is assumed to be 6 dB or so [The above simulations assumed a fixed SNR (=10 dB) situation to permit a comprehensive evaluation. When we assume a total transmission power of 10 dBm, 0 dBi antenna gain, −174 dBm/Hz noise density and free space propagation, average reception SNR in the SISO case is about 9 dB in the simulation environment. Of course, the actual value depends on system parameters and deployment environment and the received signal strength significantly impacts the capability of the proposed CSI estimation scheme. To discuss the application region, dB or so . As seenAs a result, our proposal can eliminate the link budget shortfall for CSI estimation in the millimeter waveband. It should be noted that assuming OFDM, timing extraction for fast Fourier transform (FFT) windowing for the CSI estimation on the reduced subcarrier is a critical issue since it is performed in the time domain, i.e., the gain provided by the reduced number of training subcarriers cannot be obtained. Meanwhile, millimeter wave small cells can be supported by control signaling from macro cell using micro wave band . It willThis paper verified the effectiveness of 1st eigenmode transmission in multiuser massive MIMO assuming LoS dominant channels in the millimeter wave band. Because of the 1st eigenmode’s robustness and high-gain property, quite a large number of UEs can be spatially multiplexed; it is effective in enhancing the system throughput rather than UE throughput. Additionally, we proposed simplified beamforming with highly efficient CSI estimation in order to ease heavy computation complexity imposed by 1st eigenmode transmission. It can also suppress the additive noise effect and thus improve CSI estimation accuracy. Our approximate approach achieved performance comparable to that of rigorously applied 1st eigenmode transmission.


Background MET expression and activation appear to be important for initiation and progression of triple-negative breast cancer. Tivantinib (ARQ 197) is an orally administered agent that targets MET, although recent preclinical data suggests the agent may have mechanisms of action that are independent of MET signaling. We conducted a phase 2 study of tivantinib monotherapy in patients with metastatic triple-negative breast cancer. Methods Patients with metastatic triple-negative breast cancer who had received 1 to 3 prior lines of chemotherapy in the metastatic setting were enrolled into this two-stage, single arm phase 2 study. Treatment consisted of twice daily oral dosing of tivantinib (360 mg po bid) during a 21-day cycle. Patients underwent restaging scans at 6 weeks, and then every 9 weeks. Tumor biomarkers that might predict response to tivantinib were explored. Results 22 patients were enrolled. The overall response rate was 5 % (95 % CI 0–25 %) and the 6-month progression-free survival (PFS) was 5 % (95 % CI 0–25 %), with one patient achieving a partial response (PR). Toxicity was minimal with only 5 grade ≥3 adverse events . Conclusion This study represents the first evaluation of tivantinib for the treatment of metastatic triple-negative breast cancer. These results suggest that single agent tivantinib is well tolerated, but did not meet prespecified statistical targets for efficacy. Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in American women . DespiteMET is a membrane receptor normally expressed on cells of epithelial origin. Hepatocyte growth factor (HGF) is the only known ligand of the MET receptor. Upon HGF stimulation, MET induces several biologic responses that lead to invasive growth. MET overexpression, with or without gene amplification, has been reported in a variety of malignancies, including breast, colorectal, lung, gastric, and hepatocellular carcinoma , 5. ElevEarly preclinical work suggested that tivantinib (ARQ 197) binds to and inhibits MET in vitro, and phase I and II trials in a variety of malignancies demonstrated the safety and tolerability of tivantinib, and suggested promising activity of this agent –17. BecaPatients ≥18 years of age with measurable metastatic triple-negative breast cancer were eligible. Triple-negative status was defined as estrogen receptor negative (<10 % staining by immunohistochemistry (IHC)), progesterone receptor negative (<10 % staining by IHC), and HER2-negative . Patients may have received 1 to 3 prior chemotherapeutic regimens for metastatic breast cancer, and must have been off treatment for 14 days prior to initiation of study treatment. Patients must have recovered from acute toxicities of their prior treatment. Patients may have received prior radiation therapy in either the metastatic or early-stage setting, but radiation was required to be completed at least 14 days prior to initiation of study treatment. Patients were required to have an Eastern Cooperative Oncology Group (ECOG) performance status ≤2, and a projected life expectancy greater than 6 months. They were also required to provide formalin-fixed, paraffin-embedded (FFPE) tumor tissue. Key exclusion criteria included: prior receipt of a MET inhibitor; known brain metastases that are untreated, symptomatic, or require therapy to control symptoms; and QTc > 470 ms. Research was approved by local human research protections programs and institutional review boards, and studies were conducted in accordance with the Declaration of Helsinki.This was a single-arm, two-stage phase 2 study assessing the efficacy of tivantinib monotherapy in patients with metastatic triple-negative breast cancer. Tivantinib was provided by Daiichi Sankyo/ArQule through the Cooperative Research and Development Agreement (CRADA) program. Treatment consisted of oral dosing of tivantinib at 360 mg twice daily over a 21-day cycle. Patients underwent radiographic restaging at 6 weeks, and then every 9 weeks. Patients with complete or partial RECIST responses continued to receive study treatment, while those with progressive disease were taken off study. Dose reductions for toxicity occurred if patients experienced grade 3 or 4 neutropenia or non-hematologic adverse events. From the starting dose of 360 mg, doses were reduced as needed to 240 mg twice daily, 120 mg twice daily, and 120 mg once daily.The primary objective of this study was to evaluate the activity of tivantinib, as defined by 6-month progression-free survival (PFS) in patients with metastatic triple-negative breast cancer. The 6-month progression-free survival (PFS) was defined as the proportion of patients still receiving treatment at 6 months, who were alive and free of progression at this time point. Secondary objectives were to evaluate objective response based on RECIST 1.1 criteria, to evaluate MET and phospho-MET expression in archival tumor tissue, and to evaluate the incidence of MET positive circulating tumor cells (CTCs) at baseline.MET FISH probe labeled with SpectrumRed and CEP7 reference probe labeled with SpectrumGreen were purchased from Abbott Molecular . FISH was performed following standard protocols. Briefly, 5 μm tissue slides were baked overnight at 60 °C, deparaffinized, treated in 1 % sodium borohydride for 4 h and heated in pressure cooker for 20 min in citrate buffer (pH = 6). After treatment with 150 μg/ml solution of Proteinase K, slides were fixed in 1 % neutral-buffered formalin and denatured in 70 % formamide for 4 min at 72 °C. Probe was denatured for 5 min at 80 °C and incubated for 30 min at 37 °C for pre-annealing. Hybridization was carried out overnight at 37 °C; post-hybridization slide washes were carried out for 20 min in 50 % formamide/2xSSC at 45 °C, followed by 5 min wash in 1 × SSC at 45 °C. FISH signal evaluation and acquisition were performed manually using filter sets and software developed by Applied Spectral Imaging . Several fields with at least 50 tumor cells total were captured and ratio of MET to CEP7 signal numbers was calculated. Assessment of ploidy status was done by visual screening of all tumor area; cells with maximum number of signals were recorded. MET amplification was defined as a MET/CEP7 ratio ≥ 2. Samples having a MET/CEP7 ratio from 1.5 and up to 2 were defined as having relative MET gain. Samples with a MET/CEP7 ratio of 1 but with more than two copies of each probe were defined as having polysomy of chromosome 7.CTCs were enriched from 7.5 mL of a patient’s whole blood at the Circulating Tumor Cell Core Facility  using the Circulating Tumor Cell Profile Kit . Processed samples were received as cells suspended in 900 uL of buffer. An equal volume of PBS was added before tubes were spun down at 200 × g for 8 min. Supernatant was carefully removed, leaving approximately 60 uL of buffer. Cell pellets were gently resuspended and the suspension was applied on the labeled slide and allowed to dry in the vacuum desiccators at room temperature. Slides were placed in methanol at −20 °C for aging and storage.For FISH, dried slides were treated in 2 × SSC at 37 °C for 30 min, followed by 10 min of treatment with 0.002 % pepsin solution in 0.01 M HCl at 37 °C and 15 min of fixation in 1 % formalin at room temperature. Slides were dehydrated in the series of ethanols, dried and co-denatured with MET/CEP7 FISH probe  on an 80 °C plate for 2 min. Hybridization was carried out at 37 °C overnight, followed by a 0.4xSSC/0.3 % Igepal wash at 72 °C for 3 min and a 2 × SSC/0.1 % Igepal wash at room temperature for 1 min. Slides were dehydrated in the series of ethanols, and dried before application of Vectashield mounting medium with DAPI . FISH signal evaluation and acquisition were performed manually using filter sets and software developed by Applied Spectral Imaging .Available archival tumor tissue was tested centrally for MET and phospho-MET protein expression. Four-micrometer-thick sections were baked at 37 °C overnight, then deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide in methanol. For heat induced epitope retrieval, slides were placed in 10 mmol/L citrate buffer at a pH of 6.0  and then pressure cooked  at 122 °C to between 14 and 17 psi with the cycle lasting, on average, 45 min and a cool-down period of approximately 20 min. Immunostains were performed on an automated instrument . A range of titers was tested for both antibodies, and titers were calibrated using positive control staining. Primary antibodies to MET, clone SP44 , phospo-MET, clone Y1234/1235  were incubated for 40 min at room temperature. A DAKO polymer secondary antibody system was used  and incubated for 30 min in a humid chamber at room temperature. Sections were developed using 3,3-diaminobenzidine  as substrate and counterstained with Mayer hematoxylin. External positive controls were also run.MET and phospho-MET protein expression levels were recorded as percentages of positively stained tumor cells in each of the five intensity categories denoted as zero (no staining), 1+ (weak but detectable), 2+ (mildly distinct), 3+ (moderately distinct) and 4+ (strong). For each tumor, a value was derived by summing up the percentages of cells staining at each intensity, multiplied by the weighted intensity of staining: H-Score = (0 × % at 0) + (1× % at 1+) + (2× % at 2+) + (3× % at 3+). This score produces a continuous variable that ranges from 0 to 300. A positive result was set to an H-Score of 1, defined as a minimum of 1 % of tumor cells staining weakly 1+ .In the patient who achieved a RECIST response, tumor and matched germline tissue underwent DNA extraction and whole exome sequencing, using a previously described protocol . After wThis study used a Simon optimal two-stage design to control type I error at 10 % and have at least 90 % power to detect the acceptable response rate. The sample size was calculated to distinguish between a response rate of 10 versus 31 %. In the study design, 13 participants were to be enrolled in the first stage. If there were at least 2 participants who were alive and progression free at 6 months, the study would enroll another 13 participants. If there were 4 or fewer of the 26 who were alive and progression-free at 6 months, the regimen would be declared inactive. If there were 5 or more who were alive and progression-free at 6 months, the regimen would be declared active. PFS and 95 % confidence intervals (CIs) are described using Kaplan-Meier methods. The overall response is tabulated. All correlative analyses are exploratory.The study was activated in April 2012. Based on slow accrual and external findings suggesting that tivantinib may not actually inhibit MET , accrualThe baseline characteristics of the patients enrolled into the study are listed in Table In all patients, the median progression-free survival (PFS) was 1.2 months (95 % CI: 1.0–1.4), Fig. Few severe adverse events were reported by patients taking tivantinib Table . Of the Four dose reductions occurred in 2 patients due to neutropenia and fatigue. Three of four dose reductions occurred within the first two cycles. The one responder required two dose reductions, one in cycle 2 (neutropenia) and another in cycle 8 (neutropenia and fatigue).All 22 enrolled patients had adequate amounts of archival tissue for immunohistochemical (IHC) staining. Among those 22 patients, 10 had positive IHC staining for MET , while none of the patients had positive phospho-MET staining on archival tumor tissue. Of 7 patients with circulating tumor cells (CTCs) collected at baseline, 1 had 2-fold MET amplification in her CTCs as assessed by FISH, but this was not correlated with response, as the patient had progressive disease at the first 6 week restaging. Of 18 patients who had FISH testing for MET on archival tissue, 3 were amplified in <5 % of nuclei. Four patients did not have adequate amounts of archival tissue for FISH analysis. The one responder was negative for MET and phospho-MET by IHC on archival tissue, and had CTCs that were not MET-amplified. Her archival tumor tissue was amplified for MET by FISH in less than 2 % of nuclei, though the clinical significance of this is unclear  and TP53 (R110P) were identified, as well as a variant of unknown significance in the kinase domain of JAK2 (E577Q). No alterations that suggest a mechanism of response or resistance to this agent were found. Unfortunately, this patient’s baseline tumor was unevaluable using this sequencing technology, so it was not possible to determine whether any changes in mutational status led to development of tivantinib resistance.This trial represents the first study of tivantinib for the treatment of metastatic triple-negative breast cancer. Of 22 patients that were evaluated, only one patient had an objective response. The single responder did not have evidence of MET expression by IHC or clear evidence of MET amplification in her archival tumor specimen, so it is unclear if her response was due to inhibition of MET. To further understand the molecular rationale for the one responder to tivantinib in our trial, we performed whole exome sequencing on tissue from her post-progression biopsy. This analysis identified mutations in known breast cancer genes MAP2K4 and TP53, as well as a mutation in the kinase domain of JAK2. However, the patient’s archival tumor tissue sample was unevaluable, so we could not compare the mutational profile of tivantinib-sensitive versus resistant tissue.One potential explanation for the low response rate seen in this study was that the population was not enriched for patients with tumors that were high in MET overexpression. Prospective data from studies of tivantinib in the treatment of hepatocellular carcinoma found that positive MET expression (≥2+ staining in ≥50 % of tumor cells by IHC) was associated with improved overall survival (OS), PFS, and time to progression . AdditioThe low response rate in this study may also be related to the possibility that tivantinib may not be a specific inhibitor of MET. Pre-clinical work has revealed that tivantinib binds to MET in vitro, in vivo, and demonstrated disease regression in phase I, II, and III clinical trials –17. HoweWhile the reason for the low response rate seen in this study is not clear, our results indicate that though tivantinib is well tolerated, it does not have significant clinical efficacy when used as monotherapy to treat unselected, metastatic triple-negative breast cancer.


The antibiotic should be kanamycin not chloramphenicol and the correct text is: Transformed


With the increasing use of meta-analysis, duplicate publication of original research is particularly problematic. Duplicate publication can result in an inappropriate weighting of the study results. The purpose of our study was to assess the incidence and characteristics of duplicate publications in Korea, and to estimate the impact of duplicate publication on meta-analyses. The meta-analysis literature written by Korean authors was searched using the online search engines PubMed, KMbase, and KoreaMed. Duplication patterns were classified into the following 4 combinations: identical samples and identical outcomes (copy), identical samples and different outcomes (fragmentation), increased samples and identical outcomes , and decreased samples and identical outcomes (disaggregation). To estimate the multiple publication bias, we performed a meta-analysis with and without duplicated data. We estimated that 6 (6.9%) of the 86 analyzed meta-analyses included duplicate publications, and 6 of the 1,194 articles (0.5%) used in the meta-analyses were duplicate publications. In this study, duplicate publications were usually due to disaggregation and overlapping  publications. Of 6 duplicated articles, 1 was considered a copy (16.6%); 1, a fragmentation (16.6%); 2, imalas (33.3%); and 2, disaggregations (33.3%). There was an increase in the mean effect size and fail-safe number with duplicated data. Our study found only 6 instances of duplicate publication after analyzing 1,194 articles used in meta-analyses written by Korean authors. However, 6.9% of the meta-analyses included duplicate publications. Our findings suggest that meta-analyses should be interpreted cautiously, taking into account the possibility of duplicated studies. Over the past few decades, there has been an explosive increase in biomedical publications, and the practice of duplicate publication is sometimes problematic in the scientific medical community. According to the International Committee of Medical Journal Editors, duplicate (or redundant) publication can be defined as “the publication of an article that overlaps substantially with one already published in print or electronic media” . DuplicaThe incidence of duplicate publications has been measured, and varied incidences of duplicate publication have been demonstrated in previous studies. A total of 7.6% of publications in the orthopedic literature were found to have some degree of redundancy  was screened using the key word “Korea” and limited with “meta-analysis” in advanced search for “type of article”. To find meta-analyses not indexed in PubMed, the KMbase (http://kmbase.medric.or.kr) and KoreaMed (http://www.koreamed.org) were searched using “meta-analysis” as a search term. The entire search was performed by 1 librarian, and analysis of selected meta-analyses and included studies was performed independently by 4 investigators.The meta-analysis literature written by Korean authors was searched using the online search engines PubMed, KMbase, and KoreaMed. PubMed  publications. An imalas publication is an expansion of a main article through the addition of more data to produce a high-impact article. Disaggregation is the publication of several fragmented parts of a multicenter trial. Reporting the same trial more than once is unacceptable, except in the case of large, multicentre trials with numerous endpoints and following the acceptance of the editor. Generally, the title was modified, and the authors, study sample, and method underwent a minor change in the duplicate publications. All of the duplicate articles in our study were published within 2 years of the main article, and no clear cross referencing was present in the majority (83.3%). However, in 1 case in our study , there was no common authorship. In this case, the study sample, method, outcome, and the department of the authors of the 2 articles were almost identical; we therefore concluded that the articles were overlapping  publications. The use of different authors for an identical study may be referred as plagiarism, and obscures duplication. Indeed, it may be extremely difficult for reviewers to determine whether 2 papers represent duplicate publications of 1 trial or 2 separate trials when 2 articles reporting the same trial do not share a single common author.Meta-analyses in the nursing literature had the highest proportion of duplicate publications (33%), and all of the duplicate publications in this study were published before 2005. These cases of duplication might have happened accidentally, or through negligence, rather than by intention. In Korea, the concept of duplicate publications and publication ethics has been raised since 2005 following Professor Hwang’s case (Saunders and Savulescu Detection and exclusion of duplicate data is an important step in conducting a meta-analysis. Our study found an increase in the mean effect size and fail-safe number with duplicated data. This finding means that the effect and reliability was overestimated in a meta-analysis. Multiple publication bias may be an important threat to the validity of meta-analyses. Most importantly, studies with significant results are more likely to lead to multiple publications and presentations (Easterbrook et al. The reasons for duplicate publication may be varied. The authors may have no concept of duplicate publication, or may desire to approach different audience groups. However, in most cases, duplicate publication occurs to boost the author’s career advancement; in other words, the author may feel compelled to publish more for scientific achievement, which is measured by the number of published articles. To prevent duplicate publication, regular education on publication ethics and notifications to members are needed.There were some limitations in this study. The first limitation is that the judgments and categorizations were subjective in nature. Secondly, the analysis on the effect of duplicate publication on meta-analyses was performed on only 2 meta-analyses including duplicate publications. Furthermore, we did not take into account the confirmation of duplication by the original authors. However, to avoid subjective judgments, 4 reviewers independently reviewed the articles, and if necessary, consensus was reached by discussion. We believe that our findings are helpful in that they will increase the awareness of the problem of duplicate articles and reduce the incidence of duplicate publications in future.Our study found only 6 instances of duplicate publication after analyzing 1,194 articles used in meta-analyses written by Korean authors. However, 6.9% of the meta-analyses included duplicate publications. This kind of multiple publication bias will result in an accentuated positive effect of interventions. Our findings suggest that meta-analyses should be interpreted cautiously, taking into account the possibility of duplicated studies.


Infections occur frequently in critically ill patients and their management can be challenging for various reasons, including delayed diagnosis, difficulties identifying causative microorganisms, and the high prevalence of antibiotic-resistant strains. In this review, we briefly discuss the importance of early infection diagnosis, before considering in more detail some of the key issues related to antibiotic management in these patients, including controversies surrounding use of combination or monotherapy, duration of therapy, and de-escalation. Antibiotic pharmacodynamics and pharmacokinetics, notably volumes of distribution and clearance, can be altered by critical illness and can influence dosing regimens. Dosing decisions in different subgroups of patients, e.g., the obese, are also covered. We also briefly consider ventilator-associated pneumonia and the role of inhaled antibiotics. Finally, we mention antibiotics that are currently being developed and show promise for the future. Intensive care unit (ICU) patients are particularly likely to have or develop infection, in part because infection is a reason for admission and in part because of immunosuppression associated with critical illness and the large number of invasive devices used in these patients. Correct and adequate antibiotic coverage is essential but can be complex as a result of delayed identification of microorganisms, the impact of critical illness and therapy on pharmacokinetics (PK) and pharmacodynamics (PD) of antibiotics, and the high prevalence of antibiotic-resistant strains.In this review, we briefly highlight the importance of early infection diagnosis before discussing some of the key issues related to antibiotic management, including problems associated with timing, duration, and dosing. We also briefly consider ventilator-associated pneumonia (VAP), the use of inhaled antibiotics, and new antibiotic and adjunct strategies for the future. We focus on bacterial infections and issues associated with multi-drug resistance will not be covered.The diagnosis of infection in critically ill patients and identification of causative microorganisms and their antibiotic susceptibilities can be a challenge and yet early, appropriate antibiotic therapy is associated with improved outcomes , so accuIn response to this problem, more rapid microbiological identification methods are being developed, including polymerase chain reaction (PCR) and mass spectrometry with or without electrospray ionization –6. TheseIt is generally accepted that antibiotics should be administered as soon as possible once infection is identified , althougAcinetobacter baumannii  and non-fermenting Gram-negative bacteria), aerosolized antibiotics successfully eradicated the existing MDR organisms and reduced the pressure for new resistance . So. So98]. g daily) . AdverseH. influenzae, Klebsiella, and Escherichia coli [P. aeruginosa or Burkholderia.Omadacycline (an aminomethylcycline) and eravacycline (a fluorocycline) are developed from the tetracyclines and have now entered phase II clinical trials. Omadacycline is available for intravenous and oral therapy. It is effective against a large number of sensitive but also resistant Gram-positive pathogens (including vancomycin-resistant enterococci and MRSA) and against some Gram-positive pathogens, such as hia coli . EravacyPseudomonas activity, and the Pseudomonas-active ceftobiprole) are available in a number of countries, but have not been used extensively (because of a lack of data) in critically ill patients. In a recent RCT, ceftobiprole (3 × 500 mg) was compared with the combination of ceftazidime (3 × 2 g) and linezolid (2 × 600 mg) in 781 patients with nosocomial pneumonia, including 210 with VAP [Fifth-generation cephalosporins with MRSA activity  therapy has been tested against meropenem or imipenem in two RCTs in patients with intra-abdominal and urogenital infection. Non-inferiority of the new combination was demonstrated in both studies [Avibactam is a new β-lactamase inhibitor active against a large number of extended spectrum β-lactamases, including class A, some class C, and some class D β-lactamases. It is not active against the metallo-β-lactamases, but is active against  studies , 107. A P. aeruginosa in general and especially against OprD mutants of this pathogen [Two other new β-lactamase inhibitors are now being studied in phase III trials in patients with MDR enterobacteriaceae, including carbapenem-resistant strains. The boronate β-lactam inhibitor, RPX7007, combined with the new carbapenem, biapenem (RPX2003), demonstrated high bactericidal activity against carbapenem-resistant enterobacteriaceae . MK-7655pathogen .P. aeruginosa and S. aureus because of their resistance profile and Clostridium difficile because of its tendency to cause relapse or recurrence.We are facing increasing incidences of bacterial resistance for both community and nosocomial infections and there is a need for alternative, non-antibiotic, adjunctive therapeutic options to decrease antibiotic pressure. In the ICU, the most problematic microorganisms to treat remain C. difficile infections appears to be toxin-neutralizing antibodies that target both toxin A and B [C. difficile infection recurrence rates decreased threefold when oligofructose prebiotics or toxin-neutralizing antibodies were added to standard antibiotics. Recently, spores of C. difficile given by mouth were shown to be effective in stopping repeated bouts of C. difficile infection, which occur in 25–30 % of patients who suffer an initial episode of diarrhea or colitis [The only effective adjunctive therapy for  A and B . Fecal f A and B . Moreove colitis .P. aeruginosa. Repeated doses of a monoclonal antibody targeting P. aeruginosa serotype O11 as an adjunctive therapy to antibiotics in P. aeruginosa hospital-acquired pneumonia and VAP were associated with a significant resolution rate without immunogenicity [Monoclonal antibodies are probably the most promising adjunctive option for treating genicity .S. aureus, for adjunctive therapy in VAP. In a mouse sepsis model, treated animals had a significant reduction in mortality [S. aureus and MRSA strains (unpublished data). Clinical trials are ongoing in patients with VAP.Human monoclonal antibodies are also being developed that specifically bind and neutralize the alpha-toxin of ortality  and in aSome very innovative adjunctive approaches may also be beneficial in severe infections in the ICU. Pore-forming toxins (PFTs) induce lysis of host target cells by forming pores that disrupt the plasma and can cause serious complications associated with high mortality rates. About 30 % of cytotoxic bacterial proteins are PFTs, making them the largest category of virulence factors. Capturing bacterial PFTs with liposomes by mimicking membrane domains thus appears a promising approach, although it is still in pre-clinical development .Management of infection in critically ill patients is an evolving challenge, in part because of the ever-present threat of MDR strains. Alterations in PK/PD parameters in critically ill patients can complicate dosing issues, yet adequate antibiotic treatment is crucial to optimize survival rates. Therapeutic drug monitoring is likely to be more widely used in the future. Antibiotic choices and durations need to be individualized for each patient according to specific patient characteristics, disease severity, likely infecting organisms, and local resistance patterns. Although more responsible antibiotic prescribing may help reduce antibiotic pressure and development of antibiotic resistance, research needs to continue to try and identify new antibiotics and adjunctive therapies.


Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, antibiotic resistant Gram-negative bacteria are prevalent and extremely difficult to treat. Therefore, rejuvenating the therapeutic potentials of existing antibiotics represents an attractive novel strategy. Antimicrobial peptides have been of great focus recently. Newly derived synthetic lipopeptides have been shown to exhibit antimicrobial activity against bacteria and fungi.Escherichia coli and a NDM-1 producing strain.In this study, we investigated the ability of an antimicrobial peptide, PA-KKkK, to enhance the potency of currently used antibiotics against antibiotic-resistant clinical isolates of Antibiotic susceptibility was investigated by determining the minimal inhibitory concentration (MIC) using a broth dilution method. To study the combined interactions between PA-KKkK and the antibiotics, chequerboard titrations were performed. Time-kill assays were then carried out to prove the effect of synergistic activity against the tested bacterial strains. Transmembrane potential depolarisation assays and ATP levels were determined to understand the mechanism of action of PA-KKkK.E. coli. Time-kill analysis demonstrated significant synergistic activities when a low level of PA-KKkK was combined with rifampicin and colistin. PA-KKkK had a membranolytic effect on cytoplasmic membrane and in combination, decreased ATP levels of cells in a dose-dependent manner.NDM-1 producing strain was extremely resistant to all antibiotics tested. The factional inhibitory concentration index (FICI) was calculated to show the peptide synergised with rifampicin against the NDM-1 strain, while it synergised with rifampicin, colistin, ceftazidime and aztreonam against antibiotic-resistant clinical isolate of E. coli. This novel treatment regimen can have major clinical implications in our fight against Gram-negative bacterial infections.We have demonstrated that PA-KKkK acts as an antibiotic enhancer and therefore accelerates the bactericidal activity of drugs against antibiotic-resistant None declared.


This study provides a profile of 866 people living with HIV/AIDS (PLWHA) in three provinces in rural China and identifies factors associated with per-capita income in AIDS-affected households. The majority of the participants were female, married, had completed primary school, and were 30-49 years of age. Thirty percent of respondents lived in a household with at least one other HIV/AIDS patient and 15% had experienced the death of a household member due to HIV/AIDS. Therefore, health professionals should be aware of issues of grief and caregiver burnout among rural PLWHA and their families. Three-quarters of the respondents continued to work after being diagnosed with HIV/AIDS. Household per capita income was significantly higher for married individuals and those still working. Possible government and workplace policy initiatives that endeavor to increase income and mitigate the economic impact of HIV/AIDS on households are discussed. In 2007, approximately 700,000 people in China were HIV positive, with a prevalence rate in the general population of 0.05%. Although the percentages are relatively small, there are high infection rates among sub-populations in certain localities . These iThe association between HIV/AIDS and poverty has been a recurrent theme in the literature . AlthougIn looking at specific household expenditures and income trends, the serious economic impact of HIV/AIDS on households can be illustrated. In China, households containing PLWHA earn approximately 45% of the per person income of households which are unaffected by HIV/AIDS . Not onlGender plays a role in the economic impact of HIV/AIDS as a result of the division of labor between men and women determined by social and cultural norms . The ecoThe social and health consequences of HIV/AIDS disproportionately affect those who are already disadvantaged due to their gender, socio-economic status, and other related social dimensions . CompareAlthough several studies have examined factors that associated with the economic impact of HIV/AIDS on households, the majority of these studies pay disproportionate attention to the estimation or calculation of costs . This stThe survey was conducted in seven counties within the provinces of Henan, Anhui and Yunnan. The locations were selected based on their geographic location , the dominant mode of HIV/AIDS transmission , and high HIV/AIDS prevalence rates .This study used convenience sampling. In two of the provinces (Henan and Anhui), individuals with HIV/AIDS who received financial assistance targeted at PLWHA were provided with information about the study by staff who provided the social assistance. Those PLWHA who wished to participate were invited to contact study staff. In Yunnan province, that recruitment strategy was not available to us. Therefore, in Yunnan, we recruited through the health department. Staff who delivered HIV testing and health service provided the study information to PLWHA. Those PLWHA who were willing to join the research were asked to contact our study interviewers directly.Only one PLWHA who was 18 years of age or older was eligible per household. In the 30% of households in the study which contained two or more individuals who had HIV/AIDS who were eligible and available and who had volunteered to be interviewed, we had the following protocol: First, if one of the two eligible household members with HIV/AIDs was the household head, we interviewed that person. If there was no eligible PLWHA who was the household head, we interviewed the spouse of household head. If the household head and the spouse were not eligible or available, the person  who was most knowledgeable about the socio-economic status of household will be given priority to be interviewed.A total of 866 PLWHA were recruited. The participants ranged in age from 20 to 67 years old, with a slightly higher number of female participants (52%). For one-quarter of the respondents, their HIV had reached full-blown AIDS.This survey was administered between November 2006 and February 2007 in the aforementioned provinces. The study protocol was conducted and approved by the institutional review board of the Beijing Normal University. Informed consent was obtained from all participants. After an interviewer described the study, participants could refuse to participate; respondents were permitted to withdraw from the survey at any time without penalty. Careful attention was paid to the participants’ requirements regarding the time and location of interviews due to concerns regarding confidentiality and stigma. To ensure the confidentiality of participants, names and other identifying information were available only to the researchers. After informed consent was obtained, standardized interviews were administered face-to-face by trained interviewers. The interviews lasted approximately one and a half to two hours and participants were given RMB 20 as compensation for their time. This amount is the equivalent of $3 US; approximately an average day’s wage for a peasant in these regions.Household income per capita during the last year was the key variable of interest. Total household income was a sum of the following continuously measured variables: agricultural income, self-employment income, migrant income, government subsidies, relatives’ remittance, and other reported income. Income per capita was created by dividing the total household income by the number of people in the household.The following socio-demographic variables of the interviewed PLWHA in each household were recorded: gender, age, education level , marital status (married and unmarried), role of PLWHA in the household (household head and other), employment status (whether continued to work after diagnosis) and clinical stage of HIV or AIDS  were considered to have AIDS; otherwise respondents were considered to have HIV). The following household variables were measured: family size , the number of PLWHA in the household (1 vs 2 or more), and infection status of other family members.SPSS 11.5 data analysis package was used for all analyses. Household income per capita was therefore logarithmically transformed because income per capita had a skewed distribution and a multiple linear regression, which assessed household income per capita against each covariate, was run. Household income per capita and the continuous variable of age were not linearly related and therefore, age was categorized into 20-29, 30-39, 40-49, 50-59, and >60 years) and entered into the model as dummy variables. Categorical variables  were also transformed into dummy variables.The majority of the participants were female, married, had completed primary school, and were 30-49 years of age. One-quarter of the participants were in the AIDS stage of the disease. 52.1% of the participants were head of their household and over three-quarters of the respondents continued to work after diagnosis.Most households reported were composed of four to five family members. Prior to the respondents’ HIV/AIDS diagnoses, 77.8% of the households were unaffected by HIV/AIDS; 15.1% of the households had at least one family member who had died of HIV/AIDS. Thirty percent of respondents lived in a household with at least one other HIV/AIDS patient. As shown in the linear regression analysis , the logHousehold income per capita was associated with family size such that it was significantly lower in households with more family members. However due to the sharing of housing costs and other economies of scale , the actThe findings suggest that those whose disease was still in the HIV stage were significantly richer than those who had full-blown AIDS, as has been found in previous research . In our This trend of continued employment following an HIV/AIDS diagnosis contrasts with the findings of a study conducted in South Africa  where paIn this study, gender and household income per capita were not significantly related which is, again, inconsistent with existing literature. Previous studies have found that, compared to the household income of female PLWHA, the household incomes of male PLWHA can be more seriously affected by HIV/AIDS . The posSocial programs and social policies need to be created to meet the demands of caregiving. This includes the need for respite and for financial, psychological and grief support. The caregiving demand on PLWHA and their family members may be particularly acute because over one-fifth of PLWHA are aged 50 and over and 30% of households consist of two or more PLWHA. Our data indicate very limited economic resources in these households and therefore limited ability to purchase respite care. Creative social service programs could provide government employees for nursing or home care support and/or fund the hiring of private assistance and/or respite care in local communities.Psychological support and counseling service are urgently needed for households affected by HIV/AIDS. Living long-term with HIV/AIDS is a persistent challenge for PLWHA and their family members. The combination of the economic burden and stigma faced by many PLWHA underlines the importance of psychological and counseling services. Health professionals in rural China should screen individuals with HIV/AIDS to assess whether grief related support is needed. In our sample, 15% of respondents had experienced the death of a household member due to HIV/AIDS. Healthy grieving may be undermined due to the stigmatized nature of HIV/AIDS. There may also be feelings of anger at the deceased person who may have been the source of the current patient’s HIV/AIDS infection.The findings suggest that supportive employment environments for PLWHA would be beneficial in mitigating the economic impact of HIV/AIDS on households and improving the quality of life of family members. The use of HAART has transitioned HIV into a chronic disease: Workplaces must strive to accommodate this disabling condition, where possible. During our field work, some PLWHA demonstrated their strong desire to continue to work both on and off the farm. Supportive policies should be created to help PLWHA to continue working. For example, tax preferential policies for small businesses and micro-loans for necessary production materials , would be preferred by many PLWHA rather than passively receiving cash AIDS assistance. Active employment policy not only improves household economic conditions, it also raises the self-confidence of PLWHA and improves their quality of life.In addition, medical assistance is necessary for PLWHA. Current medical assistance focuses primarily on HARRT and has been very helpful to PLWHA. However, as HIV has changed into the chronic disease treatment, opportunistic infections has become an important part of HIV treatment. One possible consideration is to integrate the assessment and treatment of opportunistic infections into New Cooperative Medical System (NCMS) - the main rural medical care security system in China. Some rural regions have already piloted the integration of HIV treatment into the NCMS.Findings should be interpreted with caution due to the limitations of the study: First, our recruitment strategy in two provinces was focused on those who were receiving social assistance for PLWHA and thus our sample does not represent rural Chinese PLWHA. The sample may also not be representative of PLWHA receiving public assistance, since we have no way of assessing how many of those informed of the study declined to participate. A second limitation was use of self-report to determine previous income. A third limitation was the cross-sectional nature of the survey which limits causal inferences.Despite these limitations, this study provides valuable information regarding factors associated with per capita household income and may serve as a basis for program and policy initiatives. Future research should use representative population based samples, prospective panel studies and gather information on a greater range of factors which may influence the economic well-being of PLWHA.


We aimed to assess MET intratumoral heterogeneity and its potential impact on biomarker-based patient selection as well as potential surrogate biomarkers of MET activation.MET FISH positivity was defined by MET/CEP7 ratio ≥ 2.0 and/or MET ≥ 5.0. MET staining pattern (cytoplasmic vs. membranous) and mesenchymal markers were investigated as surrogates of MET activation.Our study included 120 patients with non-squamous Non-small-cell Lung Cancer (nsNSCLC), of which 47 were incorporated in tissue microarrays (TMA). Four morphologically distinct tumor areas were selected to assess MET heterogeneity. MET positivity by immunohistochemistry (IHC) was defined as an above-median H-score and by +2/+3 staining intensity in >50% of tumor cells (Metmab criteria). MET FISH positive and showed higher H-scores (p = 0.021). MET positivity by IHC changed in up to 40% of cases among different tumor areas, and MET amplification in 25–50%. Cytoplasmic MET staining and positivity for vimentin predicted poor survival .Median MET H-score was 140 (range 0–400) and 47.8% of patients were MET positive by Metmab criteria. Eight cases (6.8%) were MET status is highly heterogeneous among different nsNSCLC tumor areas, hindering adequate patient selection for MET-targeted therapies. MET cytoplasmic staining and vimentin might represent surrogate markers for MET activation. EGFR) [ALK) and ROS proto-oncogene 1 (ROS1) rearrangements [Despite significant advances in diagnosis and treatment, lung cancer remains the leading cause of cancer death worldwide . Non-smaEGFR)  or anaplngements predict rngements –7.MET was first identified in the late ‘80s, it is located on chromosome band 7q31 and encodes a heterodimeric transmembrane receptor with tyrosine kinase activity (RTK) [ty (RTK) . Activatty (RTK) , 11. METty (RTK) , 13.MET gene amplification in NSCLC are globally considered as adverse prognostic factors [MET gene amplification [The presence of MET protein overexpression and  factors –17. Cons factors , 19. Cli factors or whose fication . In the fication  and the fication , patientDuring the past few years, next-generation sequencing studies have revealed remarkable genetic and phenotypic differences among individual solid tumors  and alsoMET gene amplification [Finally, a recent report in patients with gastric adenocarcinoma has suggested that MET staining pattern can predict fication . Moreovefication . Thus, wThe primary aim of this study was to evaluate the potential impact of intra-tumor heterogeneity on MET evaluation and classification using different techniques and criteria. Furthermore, we sought to assess the correlation of MET status with other pathological and molecular characteristics. Finally and with exploratory purposes, we investigated potential surrogate markers of MET activity, such as MET staining pattern and the presence of mesenchymal markers by immunohistochemistry.Criteria for patient selection were non-squamous non-small cell lung carcinoma (nsNSCLC) histology and availability of tissue for the studies. A total of 124 tumor specimens from 120 patients diagnosed of nsNSCLC at our institution between 2009 and 2013 were included. Four of the 120 patients presented two different tumors, thus providing one extra specimen each. Material was available either from surgical resections, core-needle biopsies or cytological cell-blocks. Clinical data were extracted from medical records and included age, sex, smoking history, tumor disease stage and clinical follow-up information.et al. [Based on tissue availability, 47 of the patients were selected to construct tissue microarrays (TMAs) as outlined by Kononen et al. . First, MET fluorescence in situ hybridization (FISH) evaluation was performed on unstained formalin-fixed and paraffin-embedded(FFPE) tissue sections from the whole tumor and the TMA samples, as previously described [MET/CEP7 probe cocktail  according to manufacturer's instructions. A minimum of fifty non-overlapping cells with hybridization signals were examined for each case with a BX51 fluorescence microscope  and using the Cytovysion software . Tumors with MET/CEP7 ratio ≥ 2.0 (named “truly amplified”) and/or MET ≥ 5.0 copies (named “high polysomy”) were considered MET FISH positive [MET gains -defined as a mean copy number ≥ 2.5 copies in at least 10% of analysed nuclei- were also recorded.escribed , using apositive , 31. METMET immunohistochemistry (IHC) evaluation was performed using anti-total c-MET (SP44) Rabbit Monoclonal as a primary antibody  and revealed using an Anti-RbOmniMap DAB Detection Kit . The staining was carried out according to the manufacturer's protocol on a Discovery XT platform . The primary antibody was incubated for 60 minutes. IHC staining was evaluated by one pathologist using two different methods. The first one was an H-score, as initially described to evaluate EGFR expression . BrieflyE-cadherin and vimentin as EMT immunohistochemical markers were evaluated semiquantitatively . Anti-huMET FISH with histopathological variables. These associations were analysed using Chi-square or two-sample T-tests as necessary. Heterogeneity between different cores  was assessed using Kappa agreement index for categorical variables (i.e. FISH categories) and intraclass correlation coefficient for continuous variables (i.e. MET H-score).All 196 TMA cores were considered and analysed as individual cases to study the association between MET IHC and Survival analyses were only performed in those patients included in the TMAs as this was a more homogeneous population, being all surgically treated patients with early stage disease. Survival curves were obtained with the Kaplan-Meier method and significance of the differences in outcome was evaluated with the Cox regression test. Statistical analysis was carried out with SPSS version 13.0 . Data and statistical analysis reported are fully compliant with the REMARK guidelines .Patients' median age was 66 years, 69% were males and 52% were current smokers. Forty percent of the study population had stage I disease and 85% were adenocarcinomas Table . Most ofKRAS and EGFR mutations were found in 21% and 12% of the samples, respectively, whereas 2% of the cases presented ALK rearrangements. Patients included in the TMA study had similar characteristics, but with a higher proportion of patients with stage I disease (60%) and EGFR mutated cases (21%).Mutational data was available for more than 90% of the cases. MET status by FISH was evaluable in 117 out of 124 tumors (94.4%). We found eight MET positive cases . Four of these cases exhibited a MET/CEP7 ratio ≥ 2 (truly amplified) and the remaining four had five or more copies of the MET gene (high polysomy). MET gain was identified in 73 cases (62.4%), being most of them polysomic for chromosome 7 (n = 60)  (data not shown). Different FISH patterns are illustrated in MET IHC was assessable in 115 out of 124 tumors (92.7%). According to MetMab criteria, 55 cases  were classified as MET high, and 60 cases  as MET low Table .MET FISH. As expected, histological pattern and grade showed a highly heterogeneous distribution among different cores . When MET IHC status was analyzed considering the H-score as a continuous variable, intraclass correlation coefficient (ICC) was 0.47 between cores A and B. When all four cores (A to D) were included in the analysis, ICC was 0.57. When cases were classified using MetMab criteria and divided into MET high and Met low, comparison of core A with the remaining three cores (B to D) revealed differences in classification in approximately 20–40% of the cases  were assessable for histology, 176 (89.8%) for grade, 184 (93.9%) for MET IHC and 180 (91.8%) for MET FISH analysis, when evaluated as a categorical variable , Kappa agreement index between cores A and B was 0.35. Regarding MET gain as a continuous variable, ICC between the four cores was 0.58. Among the three MET FISH positive cases found in the TMA population, four out of the 12 cores represented were FISH negative. Moreover, none of the cases was considered positive in all four cores   with an H-score of 400 and MET high by MetMab criteria, whereas the remaining three cases had an intermediate-level of MET amplification (MET/CEP7 ratio ≥ 2.2- < 5), of which one was classified as MET high and the remaining two as MET low by IHC. Discordance between IHC and FISH is illustrated in Figure = 0.021) . Among tariables . Applyin , whereas non-smoking was associated with a membranous staining pattern (p = 0.042)  corresponding to 11 patients showed a predominantly cytoplasmic staining. Heterogeneity of MET staining pattern among different tumor cores was also observed Figure . Three pp = 0.042). Also, tumors showing mesenchymal features had significantly lower H-scores (p = 0.027), whereas the opposite occurred for E-cadherin positive tumors, which had significantly higher H-scores (p = 0.003)  showed a mesenchymal phenotype (strong vimentin staining). All of these patients had a smoking history (five of them were current smokers and the remaining three were former smokers). Interestingly, the presence of a mesenchymal phenotype was associated with a predominantly cytoplasmic MET staining . Interestingly, patients with tumors showing either a predominantly cytoplasmic Met staining or expression of mesenchymal features (i.e. vimentin positivity) had shorter survival times, and these differences were statistically significant . Survival curves are illustrated in Figure Median follow-up time was 73.2 months and median survival time was not reached. One-, two- and three-year survival rates were 93.7%, 80.6% and 73.1%, respectively. Patients whose tumor Met H-score values were below the median had shorter survival times when compared with patients with above-median values, but thiswas not statistically significant  [The difficulty of finding the correct predictive biomarker for MET-targeted therapies may explain, at least in part, the lack of success of the two largest trials testing MET inhibitors combined with Erlotinib in NSCLC patients. The MARQUEE trial  selected = 0.03) .MET gains or gene amplification. In the MARQUEE study, no statistically significant differences were observed in overall survival between MET amplified and non-amplified cases  [EGFR wild-type and MET FISH positive [p = 0.06) [MET amplification. Those patients with a MET/CEP7 ratio of ≥ 5.0 showed significantly better outcomes [MET status by FISH is less heterogeneous than MET IHC remains to be determined, as small numbers in our study (only three FISH positive cases in the TMA cohort) prevent us from drawing any robust conclusions.Another strategy for the development of MET inhibitors in NSCLC relies on patient selection based on  = 0.34) . Converspositive  patients = 0.06) . Recentloutcomes . AlthougClassically, it has been accepted that, after activation at the cell membrane, tyrosine-kinasereceptors (RTK) are internalized and degraded or recycled back to the membrane. However, during the last decade, preclinical evidence has emerged that highlights the role of receptor endocytosis and intracellular trafficking in RTK-mediated signaling –49. In aFinally, our evaluation of potential surrogate markers for MET activation revealed interesting findings. Predominant cytoplasmic staining, which may translate MET pathway activation, was associated with a mesenchymal phenotype, which in turn can also be derived from MET HGF-dependent activation . AlthougIn conclusion, our study shows that MET status is highly heterogeneous within nsNSCLC tumors. This notion challenges current techniques and criteria for selecting patients for MET-targeted therapies. Further studies are needed to accurately detect patients with MET-driven tumors.