528 S.E.2d 166 (2000)
32 Va. App. 395
Hurcus Jerome WILLIAMS
v.
COMMONWEALTH of Virginia.
Record No. 2982-97-1.
Court of Appeals of Virginia.
May 2, 2000.
*167 Lenita J. Ellis, Norfolk, for appellant.
Steven A. Witmer, Assistant Attorney General (Mark L. Earley, Attorney General, on brief), for appellee.
Present: FITZPATRICK, C.J., BENTON, COLEMAN, ELDER, BRAY, ANNUNZIATA and BUMGARDNER, JJ., and OVERTON, Senior Judge.

UPON A REHEARING EN BANC
BENTON, Judge.
The trial judge convicted Hurcus Jerome Williams of capital murder, robbery, and two counts of using a firearm in the commission of a felony. Williams contends the trial judge committed reversible error by (1) admitting in evidence a non-testifying accomplice's out-of-court confession to police and (2) refusing to provide the accomplice's confession to Williams' counsel before ruling on its admissibility. A panel of this Court, with one judge dissenting, affirmed the convictions. See Williams v. Commonwealth, 30 Va.App. 378, 517 S.E.2d 246 (1999). Upon *168 rehearing en banc, we reverse the convictions and remand for a new trial.

I.
The grand jury indicted Williams for killing Vareck Griffin in the commission of robbery, see Code § 18.2-31 (capital murder), robbery, see Code § 18.2-58, using a firearm in the commission of capital murder, see Code § 18.2-53.1, and using a firearm in the commission of robbery. See id. The evidence at trial proved that early in the morning on December 1, 1996, someone shot and killed Vareck Griffin in Norfolk, Virginia, in an apartment where Griffin and others sold "crack" cocaine. One of the two men who operated the cocaine enterprise testified that he left Griffin alone in the apartment to sell cocaine to any potential customers.
Five and one-half months after Griffin was killed, the police arrested Damyel Harris for "more than 11 charges." The detective who interrogated Harris was investigating a homicide unrelated to Griffin's killing and had been searching for Harris in connection with that unrelated homicide. The detective testified that "[w]hen we started asking [Harris] about the [unrelated] homicide, [Harris] started talking about a different homicide and started giving details, and that's when [the detective] ... realized that [Harris] wasn't talking about [the unrelated homicide] but another homicide." During the interrogation, Harris gave an extensive statement in which he confessed to participating in the robbery of Griffin and said Williams killed Griffin.
The Commonwealth called Harris as its witness. When Harris asserted his Fifth Amendment privilege and refused to testify, the trial judge admitted in evidence Harris' confession and overruled Williams' objection that use of the confession violated his Sixth Amendment right to confront witnesses against him. In addition to Harris' confession, the Commonwealth proved through the testimony of three witnesses that Williams had made statements, which the Commonwealth contends linked Williams to the murder and robbery of Griffin.
At the conclusion of the evidence, the trial judge convicted Williams of capital murder, robbery, and using a firearm in the commission of both felonies. Williams contends on appeal that the trial judge committed reversible error by admitting Harris' confession in evidence and by refusing to provide Harris' confession to his counsel for review before the judge ruled on its admissibility. In its brief on this rehearing en banc, the Commonwealth concedes that the trial judge's admission of Harris' confession was error, see Lilly v. Virginia, 527 U.S. 116, 139, 119 S.Ct. 1887, 1901, 144 L.Ed.2d 117 (1999) (holding that "[t]he admission of the untested confession of [a codefendant] violated petitioner's Confrontation Clause rights"), and that the judge also erred in refusing to permit Williams' counsel to see the confession before the judge ruled on its admissibility. The Commonwealth contends, however, that both errors were harmless. We hold that the admission of Harris' confession was not harmless error. Because the trial judge's error in refusing to permit Williams' counsel to see Harris' confession will not recur if Williams is retried, we need not address whether that error was harmless.

II.
Although Confrontation Clause error is of constitutional magnitude, it is subject to harmless error analysis. See Delaware v. Van Arsdall, 475 U.S. 673, 684, 106 S.Ct. 1431, 1438, 89 L.Ed.2d 674 (1986). Constitutional error is harmless, however, only if "the beneficiary of the constitutional error ... prove[s] beyond a reasonable doubt that the error complained of did not contribute to the verdict obtained." Chapman v. California, 386 U.S. 18, 24, 87 S.Ct. 824, 828, 17 L.Ed.2d 705 (1967). "The test, therefore, is not whether laying aside the erroneously admitted evidence there was other evidence sufficient to convict beyond a reasonable doubt ..., but, more stringently, `whether there is a reasonable possibility that the evidence complained of might have contributed to the conviction.'" Thompson v. Leeke, 756 F.2d 314, 316 (4th Cir.1985) (citation omitted). In other words, even if "the other evidence amply supports the ... verdicts, [error is not harmless when] the disputed testimony may well have affected the ... *169 decision." Cartera v. Commonwealth, 219 Va. 516, 519, 248 S.E.2d 784, 786 (1978).
An "emphasis and perhaps overemphasis, upon the [concept] of `overwhelming evidence'" has the effect of clouding the relevant question "`whether there is a reasonable possibility that the evidence complained of might have contributed to the conviction.'" Chapman, 386 U.S. at 23, 87 S.Ct. at 827 (footnote and citations omitted). As the Supreme Court has stated, "[t]he correct inquiry is whether, assuming that the damaging potential of the [evidence] were fully realized, a reviewing court might nonetheless say that the error was harmless beyond a reasonable doubt." Van Arsdall, 475 U.S. at 684, 106 S.Ct. at 1438; see also Olden v. Kentucky, 488 U.S. 227, 232, 109 S.Ct. 480, 483, 102 L.Ed.2d 513 (1988). Thus, "a harmless error analysis ... [is not] simply a sufficiency of the evidence analysis." Hooker v. Commonwealth, 14 Va.App. 454, 458, 418 S.E.2d 343, 345 (1992).
Whether such an error is harmless in a particular case depends upon a host of factors.... These factors include the importance of the witness' testimony in the prosecution's case, whether the testimony was cumulative, the presence or absence of evidence corroborating or contradicting the testimony of the witness on material points, the extent of cross-examination otherwise permitted, and, of course, the overall strength of the prosecution's case.
Van Arsdall, 475 U.S. at 684, 106 S.Ct. at 1438. Thus, "Van Arsdall requires a court to consider several specific factorsmore than just sufficiency of the evidencewhen deciding whether the Confrontation Clause error was harmless beyond a reasonable doubt." Howard v. Gavin, 810 F.Supp. 1269, 1275 (S.D.Ga.1993).
Here we apply a more stringent standard against the government. In a sufficiency analysis we review the evidence to determine whether the government has satisfied the minimum required in order for a reasonable juror to find guilt beyond a reasonable doubt. In performing harmless error analysis, however, the standard cuts more sharply against the government: there must be no reasonable possibility that the unconstitutionally obtained evidence contributed to the conviction.
United States v. Khoury, 901 F.2d 948, 960 (11th Cir.1990) (citing Chapman, 386 U.S. at 23, 87 S.Ct. at 827).
Among the verdicts obtained in this case was one finding Williams guilty of capital murder. To support a conviction for capital murder that is predicated upon murder in the commission of robbery, see Code § 18.2-31(4), the Commonwealth must prove beyond a reasonable doubt "a killing which takes place before, during, or after the robbery and is so closely related in time, place, and causal connection as to make the killing part of the same criminal enterprise as the robbery." George v. Commonwealth, 242 Va. 264, 277-78, 411 S.E.2d 12, 20 (1991). In other words, the evidence must "support a conclusion that the killing and theft were interdependent objects of a common criminal design." Quesinberry v. Commonwealth, 241 Va. 364, 374, 402 S.E.2d 218, 224 (1991).
An application of the Van Arsdall factors reveals that the trial judge's error in admitting Harris' confession was not harmless beyond a reasonable doubt. Harris' confession was central to the Commonwealth's case. Cf. Timbers v. Commonwealth, 28 Va. App. 187, 201, 503 S.E.2d 233, 239 (1998) (holding trial judge's error in admitting accused's admission concerning the falsity of her signature was not harmless because it was the most important evidence proving forgery); Watson v. Commonwealth, 19 Va. App. 659, 666, 454 S.E.2d 358, 362-63 (1995) (holding the influence of the inculpatory statements upon the jury was not harmless where those statements constitute the principal evidence establishing his knowledge and control of contraband). Assuming the damaging potential of Harris' confession was realized, it established the essential causal connection linking the killing to the robbery. See Satterwhite v. Texas, 486 U.S. 249, 259, 108 S.Ct. 1792, 1798-99, 100 L.Ed.2d 284 (1988) (holding that expert's testimony on "future dangerousness was critical to the death sentence" and was thus not harmless beyond a reasonable doubt).
*170 In pertinent part, Harris' confession recounted the following events:
[M]e and [Williams] met up one night.... He told me that we could do a hit, get some crack, a little bit of money.... I asked him where was it at. He said Park Place. I said no, I'm too hot out here; I been chilling out. I said, do the guys know me? So he was like, no, they don't know neither one of us. So, we went around to the house, walked up the stairs. I knocked on the door; [Williams] stood on the side.
When the guy opened the door, he had a gun in his hand. [Williams] punched the guy; the gun fell. Both of them went reaching for the gun. I grabbed a bottle and hit the guy in the head. Then [Williams] grabbed the guy. We shut the door; [Williams] grabbed the guy, took him to the back. He told me to look up under the couch and grab the dope and look on the table and grab the scales. So when I was reaching for the scales, I heard a gunshot, one gunshot. And I heard the guy say, please don't kill me. So then I went up under the couch and found the drugs. That's when I heard two more gunshots above five seconds later.
Then [Williams] came running out the door. He went straight out the door and I yelled his name. He told me not to yell his name; he kept running across the street. So I ran over there to him, went up in the hallway and he said, we'll meet ... on the back street and split up everything. So when we got around there on the back street, he counted out ninety some dollars. He said he needed the money for something, like that and told me I could take the high side of the crack and that's what we did. He told me he'll sell the scales and give me some money the next day.
Although the Commonwealth produced at trial three witnesses who testified that Williams made statements implicating himself in a killing, we cannot conclude beyond a reasonable doubt that the improperly admitted testimony did not affect the verdict. Indeed, we cannot even conclude that the testimony of those witnesses and the other circumstantial evidence were sufficient, without Harris' statement, to prove that Williams killed Griffin, that a robbery occurred, or that a robbery bore the essential causal connection to the killing. As to those elements, Harris' confession was crucial, indeed essential, to the Commonwealth's case. "There was no physical evidence such as fingerprints... or human blood evidence to link [Williams to the events that occurred in the apartment]." Lilly v. Commonwealth, 258 Va. 548, 552, 523 S.E.2d 208, 209 (1999).
Harris' confession was not cumulative of the other evidence the Commonwealth presented at trial. Jesse Keene testified that Williams informed him that he had been "lay[ing] low" because, when Williams and Harris went to the apartment, "the other kid that was there got killed in the process." Keene further testified as follows:
Q. Did [Williams] tell you anything else about what happened at [the apartment]?
A. Other than that, no.
Q. Did he tell you how the kid that was at [the apartment] got killed?
A. No.
Keene's testimony clearly is insufficient to prove that Williams shot Griffin or that a robbery occurred. Although the trier of fact arguably might infer from Keene's testimony that Griffin was shot while Williams and Harris were at the apartment and that either Williams or Harris may have committed the murder, it is impossible to determine from Keene's testimony who in fact shot Griffin. It is just as likely that Williams was "lay[ing] low" because he was present at the apartment when Harris or someone else shot Griffin (and, therefore, feared being accused of the murder) as that Williams was "lay[ing] low" because he shot Griffin.
Jason Carter testified that while he and Williams were in jail, he overheard Williams tell another prisoner that Williams was in the apartment when Griffin was shot and killed. In pertinent part, he testified as follows:
Q. What did you hear [Williams] say?
A. I heard him say that he went into the house and ... [Griffin] was the only one there, and he went for the gun and he tussled

*171 Q. Who had the gun originally?
A. [Griffin].
Q. Who went for the gun?
A. [Williams].
Q. Did he say what happened after that?
A. He said he got the gun from him and he was shot.
Q. Who was shot?
A. [Griffin].
Q. Did he say who shot [Griffin]?
A. No. He just say he grabbed the gun from him. I was believing that he had shot him because by the way he was talking.
Q. Did he say why [Griffin] got shot?
A. I guess because he tried to give up the fight.
Q. Did he say why he went to the house where [Griffin] was?
A. No.
Q. Did he say if he went alone or with someone else?
A. It was with someone else.
This testimony does not clearly establish that Williams shot Griffin and clearly fails to prove a robbery occurred. Moreover, because Carter was Griffin's cousin, and Carter testified to being "close" to Griffin, we cannot conclude that the trier of fact would nonetheless have believed Carter's testimony, despite his bias, had Harris' statement been excluded.
Thomas Liggins testified that he met Williams in jail. Liggins admitted to having been convicted of two felonies and of making a false statement. He also testified that other charges were pending against him at the time of Williams' trial. Admitting further that, in return for his testimony, the Commonwealth agreed to a suspended sentence on his pending felony charge and to other consideration, Liggins testified as follows:
Q. What did [Williams] tell you?
A. That he shot somebody.
Q. Did he tell you where he shot this person?
A. No.
Q. Did he tell you who it was?
A. No.
Q. Did he tell you the circumstances of how he shot this person?
A. No. He just say he ain't had no mask on, he had to shoot him....
Q. Did he tell you what was happening when he shot the person?
A. No.
Q. Did he tell you whether he was alone or with someone else when he shot this person?
A. He was with someone.
Q. Did he tell you if it was a man or a woman that he shot?
A. He ain't say.
Q. Did he say whether he did the shooting or the other person did the shooting?
A. He said he did the shooting.
Q. Did he tell you what the other person did?
A. Yeah.
Q. What did he say the other person did?
A. He said he hit him with a bottle.
Q. Did he tell you whether this happened in a house or outside on the street?
A. No, he ain't say.
Q. Did he tell you about any other crimes besides the shooting?
A. No.
Even if the trier of fact believed Liggins, his testimony established neither that Williams was in the apartment where Griffin was killed nor that a robbery occurred.
Simply put, we cannot conclude beyond a reasonable doubt that, had the trial judge not considered Harris' confession, he would nonetheless have convicted Williams of the murder of Griffin in the commission of robbery. The effect of Harris' confession, inculpating Williams, on a trier of fact's view of the entirety of the evidence cannot be overstated. When the issue is "the potential for harm caused by the erroneous admission of evidence which tends to support the [fact finder's] credibility determination[,] ... we must presume that such evidence had the potential to influence the [fact finder] into accepting the properly admitted evidence as more credible and, thus, to taint the [fact *172 finder's] determination of the facts." Lilly, 258 Va. at 553, 523 S.E.2d at 210. Evidence which the trier of fact would normally hold as not credible or inconclusive can suddenly become convincing when corroborated by the accusatory confession of one who was intimately involved in the execution of the crime. Indeed, during the Commonwealth's closing argument, the trial judge noted that after he heard the testimony of Keene, Carter, and Liggins, which occurred before the Commonwealth introduced Harris' confession, the trial judge had written in his notes, "no robbery proved yet." The prejudicial impact of Harris' confession clearly was significant and undermines confidence in the verdict. In view of the generalized nature of the other testimony, we cannot conclude "beyond a reasonable doubt that the [improperly admitted confession] did not contribute to the verdict obtained." Chapman, 386 U.S. at 24, 87 S.Ct. at 827. Thus, the Commonwealth has failed to prove beyond a reasonable doubt that the improperly admitted evidence was harmless. Therefore, we reverse the convictions and remand for retrial if the Commonwealth be so advised.
Reversed and remanded.


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Character actor Jon Polito, best known for his roles in Coen Brothers films, including “The Big Lebowski” and “Miller’s Crossing,” died of cancer on Thursday at City of Hope Hospital in Los Angeles. He was 65.

His managers made the announcement on Friday morning after John McNaughton, his friend and a film director, first shared the news on Facebook. “Very sad to learn that my dear friend and collaborator, Jon Polito, has passed away,” McNaughton wrote.

“He appeared in over 100 films, countless TV episodes, and on Broadway,” he added. “Jon was a born actor and will be deeply missed by his legion of friends, fans, family, and of course his long time partner, Darryl Armbruster, to whom I send my condolences. R.I.P. old pal.”

According to TMZ, the actor died after being taken off life support. He reportedly fell into to a coma on Sunday and was diagnosed with multiple myeloma in 2010. TMZ also reports that he suffered from arthritis and an infection from a recent surgery.

Born on Dec. 29, 1950, Polito studied acting at the University of the Arts in Philadelphia. He made his TV debut in the 1981 NBC miniseries “The Gangster Chronicles,” playing Thomas “Three Finger Brown” Lucchese, after first breaking out on stage.

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He was an original cast member on the NBC crime drama “Homicide: Life on the Street,” and appeared in “Seinfeld” (as the super who wanted to evict Newman), “Modern Family,” “It’s Always Sunny in Philadelphia,” and “Major Crimes.”

Polito also appeared on Broadway in “American Buffalo” (1977), “Curse of the Aching Heart” (1982), “Total Abandon” (1983), and a revival of “Death of a Salesman” (1984).

He is survived by his husband, Darryl Armbruster.


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Background {#Sec1}
==========

Acute intermittent porphyria (AIP, MIM \# 176000) is an autosomal dominant disorder caused by a deficiency of hydroxymethylbilane synthase (HMBS; EC 2.5.1.61), which catalyzes the third step of haem biosynthesis. AIP is the most common acute porphyria, with a prevalence of 5.9 cases/million inhabitants in Europe as a whole and 6.3 cases/million inhabitants in Spain \[[@CR1]\].

The main clinical manifestations are episodic acute neurovisceral attacks characterized by abdominal pain, vomiting, tachycardia, hypertension and dark urine.

AIP is a low-penetrant disease, and generally 10--20% of AIP carriers develop acute attacks \[[@CR2]--[@CR4]\], known as manifest AIP (MAIP), as opposed to latent AIP carriers (LAIP) with no attacks. However, studies based on the frequency of pathogenic variants in *HMBS* gene in the general population suggest significantly lower penetrance (\< 1%) \[[@CR5], [@CR6]\]. On the other hand, higher penetrance has been associated with specific mutations, such as R173W and W198X. \[[@CR7]\].

Acute attacks are more frequent in women and rarely occur before puberty \[[@CR8], [@CR9]\]. Such acute attacks are often triggered by precipitating factors, which include several drugs, alcohol, steroid hormones, infection or fasting. Genetic background might be involved in variations in penetrance, although the underlying genes involved are still unknown \[[@CR10]\].

In the liver, free haem exerts negative feedback regulation of ubiquitous 5-aminolevulinic acid synthase (ALAS1; EC 2.3.1.37), the rate-limiting enzyme in haem biosynthesis \[[@CR11]--[@CR13]\]. Acute attacks occur when hepatic haem synthesis is overstimulated. This causes overproduction of the porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), which have been associated with clinical manifestation of AIP, and increases in urinary excretion. After acute attack, ALA and PBG gradually decrease, although levels may remain high for time ranging from weeks to years \[[@CR14], [@CR15]\].

Drug-metabolizing cytochrome P450 enzymes (CYP) constitute the main hepatic haemoproteins \[[@CR16]\]. CYP and ALAS1 synthesis is coordinated, while several drugs are transcriptional inducers of both genes \[[@CR17], [@CR18]\]. Since the turnover of CYP is a determining factor in the hepatic induction of haem synthesis, it might influence the penetrance of hepatic porphyrias.

Inter-individual differences in drug metabolism are common, in part due to inherited polymorphisms in *CYP* genes \[[@CR19]\]. A high frequency of two polymorphisms in *CYP1A2* and *CYP1A1* genes has been reported in porphyria cutanea tarda, both polymorphisms being associated with increased enzymatic activity \[[@CR20], [@CR21]\]. In addition, non-functional *CYP2D6\*3* and *\*4* alleles seemed to be less frequent in AIP carriers compared to a control population \[[@CR22]\].

We hypothesized that *CYP* genes might act as modifiers in AIP, and that specific alleles would constitute susceptibility factors for developing acute attacks. Taking advantage of our genetically highly homogeneous population (most AIP cases carry the founder mutation c.669_698del30 in the *HMBS* gene) \[[@CR23]\], we aimed to identify AIP penetrance modifying *CYP* genes that act as risk factors for developing acute attack. The goal of this study was to determine AIP prevalence and penetrance in our region associated with the *HMBS* founder mutation and the frequency of common defective alleles that lead to a disturbance in main hepatic CYP enzymes \[[@CR24]\], as well as to analyse their relationship with the occurrence of acute attacks in AIP. With this aim, *CYP2C9\*2*, *\*3*; *CYP2C19\*2*; *CYP2D \*4*, *\*5*; *CYP3A4\*1B* and *CYP3A5\*3* alleles were studied in a group of AIP carriers with a high prevalence of the aforementioned founder mutation.

Methods {#Sec2}
=======

Patients and clinical assessment {#Sec3}
--------------------------------

Fifty Spanish AIP genetic carriers, from 21 different families in the Region of Murcia (Southern Spain), were included in the study. Informed consent approved by the Research Ethics Board of the Hospital Clínico Universitario Virgen de la Arrixaca was obtained from all patients. Most of them (78%) carried the founder pathogenic variant NC_000011.9 (NM_000190.3):c.669_698del30 in *HMBS* gene responsible for AIP.

To determine the occurrence of acute attacks (MAIP frequency), patients were interviewed and clinical records were revised based on a systematic follow-up of AIP carriers currently carried out by the Medical Genetics Section of our hospital, which is the referral unit in our province. Patients who had history of at least one acute attack with a typical porphyrin precursor excretion profile, requiring hospitalization and treatment with haemin were classified as MAIP.

CYP genotyping {#Sec4}
--------------

DNA was isolated from blood samples with the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). Genotyping of *CYP2C9\*2* (rs1799853; NC_000010.10:g.96702047C \> T), *CYP2C9\*3* (rs1057910; NC_000010.10:g.96741053A \> C); *CYP2C19\*2* (rs4244285; NC_000010.10:g.96541616G \> A); *CYP2D6\*4* (rs3892097; NC_000022.10:g.42524947C \> T), *CYP3A4\*1B* (rs2740574; NC_000007.13:g.99382096C \> T) and *CYP3A5\*3* (rs776746; NC_000007.13:g.99270539C \> T) alleles was performed with TaqMan® Drug Metabolism Genotyping Assays (Applied Biosystems, Foster City, USA). *CYP2D6\*5* allele, entailing *CYP2D6* gene deletion, was analysed with TaqMan® Copy Number Assay (Applied Biosystems). All assays were performed with available commercial pre-designed kits. Assays were run on an ABI® 7500 Fast Real-Time PCR System (Applied Biosystems) and analysed with 7500 Software and CopyCaller (Applied Biosystems).

Individuals carrying none of the analysed defective alleles were predicted to carry normal functional allele since other defective alternative alleles are rare in our population, such as *CYP2C19\*3* which may be frequent in Asian populations as opposed to Caucasian.

Biochemical analyses {#Sec5}
--------------------

First morning urine samples were collected at a symptoms-free stage, at least after 6 months of an acute attack. Samples were available only in 45 patients. Urinary creatinine was analysed with a commercial kit (CREJ2) based on the Jaffé method in a Roche Cobas c system (Roche, Mannheim, Germany). ALA and PBG were measured by spectrophotometry after column chromatography with a commercial kit (code 11017, BioSystems S.A., Barcelona, Spain) following manufacturer's instructions. The results were normalized to urinary creatinine.

Statistical analysis {#Sec6}
--------------------

Continuous variables were summarised with means and standard errors, while qualitative variables were expressed as proportions. A logistic regression model, adjusted for sex and age, was used to test the association between *CYP* genotype and MAIP frequency. To test the association between *CYP* genotype and urinary ALA and PBG levels, a multiple lineal regression analysis was used. This regression model was adjusted for sex, age and history of acute attacks (MAIP) to avoid confusion in the results obtained, since ALA and PBG may remain elevated for many years after an acute attack. A simple test to compare proportions was made to analyse differences in allelic frequencies between MAIP and LAIP. Data were analysed using R software package (3.4.1. version).

Results {#Sec7}
=======

Fifty AIP carriers aged between 16 and 77 years (44 years of mean age), 56% women, were analysed (Table [1](#Tab1){ref-type="table"}). 78% carried the known founder pathogenic variant c.669_698del30 in *HMBS* gene. General penetrance was 52% (frequency of MAIP). Penetrance associated with the founder pathogenic variant was 48.7% (19/39 patients), which is similar to that associated with the pathogenic variant c.76C \> T (50%), the second most frequent pathogenic variant in the studied cohort. Furthermore, based on population the census in our province (1,470,273 inhabitants) and the frequency of MAIP described in this study, the estimated symptomatic AIP prevalence was at least 17.7 cases/million inhabitants.Table 1Characteristics of AIP carriersTotal (*n* = 50)LAIP (*n* = 24; 48%)MAIP (*n* = 26; 52%)Age, years44 ± 15.742 ± 15.847 ± 14.9Women28 (56%)10 (42%)18 (69%)*HMBS* pathogenic variant c.669_698del30, p.(Glu223_Leu232)39 (78%)20 (83%)19 (73%) c.76C \> T, p.Arg26Cys8 (16%)4 (17%)4 (15%) c.275 T \> C, p.(Leu92Pro)1 (2%)--1 (4%) c.750delA, p.(Glu250GlufsTer4)2 (4%)--2 (8%)*LAIP*: Latent AIP; *MAIP*: Manifest AIP. Age shown as mean and standard deviation; sex and *HMBS* variants distribution shown as number of individuals and percentage. *HMBS* variants are referenced to NC_000011.9 (NM_000190.3)

Allele frequencies are shown in Table [2](#Tab2){ref-type="table"} and genotype distributions are summarized in Table [3](#Tab3){ref-type="table"}. All genotype frequencies met the Hardy-Weinberg equilibrium. The frequency of defective *CYP2D6* alleles was 3.5 times higher in LAIP than in MAIP. MAIP was less frequent in defective allele carriers of *CYP2D6* gene (*CYP2D6\*4* or *\*5* alleles), so the possibility that a patient had MAIP was reduced by 80% on average for each additional defective allele (*\*4* or *\*5*) in *CYP2D6* gene (OR 0.2; CI 95% 0.04--0.81; *p* value 0.037).Table 2*CYP* allelic frequenciesGeneAlleleTotal AIPLAIPMAIP*P* value*CYP2C9\*2*0.280.330.230.255*\*3*0.010.020*CYP2C19\*2*0.150.130.170.695*CYP2D6\*4*0.120.190.060.052*\*5*0.010.020*CYP3A4\*1B*0.030.020.041.000CYP3A5*\*3*0.950.960.940.913*MAIP* manifest AIP, *LAIP* latent AIPTable 3*Cytochrome P450* genotype distributions and MAIP frequency by genotypeGeneGenotypeGenotype distributionMAIP frequencyN%%ORCI 95%*P* value*CYP2C9*two normal alleles275455.60.450.17--1.090.092one allele *\*2* or *\*3*173458.8two alleles *\*2* or *\*3*61216.7*CYP2C19*two normal alleles357048.61.750.49--6.810.398one allele *\*2*153060.0two alleles *\*2*00--*CYP2D6*two normal alleles387660.50.20.04--0.810.037one allele *\*4* or *\*5*112227.3two alleles *\*4* or *\*5*120*CYP3A4*two normal alleles479451.11.940.14--49.630.629one allele *\*1B*3666.7two alleles *\*1B*00--*CYP3A5*two normal alleles00--0.670.07--5.020.697one allele *\*3*51060.0two alleles *\*3*459051.1*OR* Odds ratio adjusted for age and sex*, CI 95%* 95% confidence interval for OR, *MAIP* manifest AIP

There was also slight evidence that MAIP was reduced by each additional defective allele (\*2 or 3\*) in *CYP2C9* gene, although not to a statistically significant extent. When a combined *CYP2C9* and *CYP2D6* genotype was considered (data not shown), only 16.7% of individuals carrying at least one defective allele in both genes had MAIP compared with 52.2% of carriers of defective alleles in either one of the genes and with 61.9% of normal allele carriers. Thus, MAIP frequency was reduced by 65% in individuals carrying at least one defective allele in both genes (OR 0.35; CI 95% 0.12--0.89; p value 0.0368). Other different *CYP* combined genotypes were not considered since there was no evidence of individual effect in acute attacks.

The urine PBG-to-creatinine ratio (Table [4](#Tab4){ref-type="table"}) tended to be lower in individuals carrying *CYP2C9\*2* or *\*3* and *CYP2D6\*4* or *\*5* alleles compared with respective normal allele carriers. However, this reduction was not attributed to the *CYP* genotype by itself but was associated with the history of acute attacks (*p* \< 0.01). There were no differences in ALA excretion according to *CYP* genotype.Table 4Urinary ALA and PBG excretion according to genotypeGeneGenotypeU-PBG/CrU-ALA/Crmean (SE)beta (CI 95%)*P* valuemean (SE)beta (CI 95%)*P* value*CYP2C9*two normal alleles12.93 (3.36)−3.82 (−9.25;-1.62)0.1638.48 (1.30)−1.26 (−8.07;5.55)0.711one allele *\*2* or *\*3*5.62 (1.88)12.85 (6.67)two alleles *\*2* or *\*3*1.87 (0.80)4.98 (0.71)*CYP2C19*two normal alleles9.12 (2.34)−1.84 (− 10.11; 6.43)0.65510.58 (3.01)−3.96 (− 14.03;6.11)0.432one allele *\*2*9.32 (4.27)6.39 (1.30)two alleles *\*2*NANA*CYP2D6*two normal alleles10.71 (2.63)−0.67 (−8.60; 7.27)0.86610.45 (2.94)−3.69 (−13.34;5.96)0.444one allele *\*4* or *\*5*5.34 (2.38)5.93 (1.05)two alleles *\*4* or *\*5*0.76 (NA)11.82 (NA)*CYP3A4*two normal alleles9.52 (2.12)−7.11 (−25.30;11.09)0.4359.67 (2.27)−6.72 (−29.08;15.65)0.547one allele *\*1B*1.88 (1.07)2.96 (1.56)two alleles *\*1B*NANA*CYP3A5*two normal allelesNA2.54 (−10.64;15.72)0.699NA5.46 (−10.63;21.54)0.497one allele *\*3*6.45 (5.00)4.33 (1.02)two alleles *\*3*9.45 (2.2)9.86 (2.38)*U-PBG/Cr* Urine PBG-to-creatinine ratio (μmol /mmol), reference range \< 1.5 μmol /mmol, *U-ALA/Cr* Urine ALA-to-creatinine ratio (μmol /mmol), reference range \< 3.8 μmol /mmol, *SE* Standard error; beta: regression analysis coefficient adjusted by age, sex and history of acute attacks; CI 95%: 95% confidence interval of beta; *NA*: Not applicable

Discussion {#Sec8}
==========

Several clinical studies have described a penetrance of around 10--20% in AIP \[[@CR2]\]. Other studies based in the prevalence of pathogenic variants in *HMBS* gene in the general populations have estimated a penetrance of below 1% \[[@CR5], [@CR6]\]. However, there are high-penetrant mutations such as R173W (50%) and W198X (44%) \[[@CR7]\], which are highly frequent in Nova Scotia (Canada) \[[@CR25]\] and Sweden \[[@CR26]\], respectively, due to a founder effect. Here, we describe the high clinical penetrance associated with the founder mutation c.669_698del30 in *HMBS* gene \[[@CR27]\], which is frequent in southern Spain. This penetrance is similar to that of the W198X mutation, which is associated with a high prevalence of AIP in Sweden (23 cases/million inhabitants). In addition, we estimated the prevalence of AIP in the Region of Murcia to be about 3 times higher than that estimated in Spain as a whole (6.3 cases/million inhabitants) \[[@CR1]\]. All the above suggest that, while low-penetrant *HMBS* mutations may be frequent in a healthy population, frequent high-penetrant mutation might be responsible for MAIP in populations with a high prevalence of AIP. High-penetrant AIP mutations may determine the genetic counselling offered to a specific population and carriers should thereby benefit from specific health care planning.

It is also of great interest to recognize how the genetic background may modulate the penetrance, making personalized genetic counselling and health care more feasible. To date, a high prevalence of specific *CYP* alleles in some types of porphyria has been described compared to that observed in a healthy population, suggesting that they might be susceptibility factors \[[@CR20]--[@CR22]\]. The present study provides further evidence that *CYP* genes may constitute penetrance-modifying factors in AIP, since *CYP2D6\*4* and *\*5* were more frequent in LAIP than in MAIP, although their allelic frequencies were similar in whole AIP carriers and the general population \[[@CR27]\]. The differential distribution of *CYP2D6\*4* and *\*5* alleles between MAIP and LAIP supports the hypothesis that *CYP2D6* may be associated with acute clinical manifestation of AIP. Accordingly, MAIP was less frequent in defective *CYP2D6\*4* and *\*5* allele carriers.

Furthermore, for the first time, the frequency of *CYP2C9\*2*, *\*3*; *CYP2C19\*2*; *CYP3A4\*1B* and *CYP3A5\*3* alleles has been determined in an AIP population, in which it was found to be similar to that of the general population and between MAIP and LAIP \[[@CR27]--[@CR29]\]. However, in our opinion, we cannot dismiss a possible relationship of these genes with AIP, which would require larger studies.

Our results suggest that *CYP2D6\*4* and *\*5* defective alleles play a protective role in the clinical onset of AIP, modulating its penetrance. AIP patients with defective *CYP2D6* alleles may be less susceptible to porphyrogenic xenobiotic intermediate metabolites. Another possible explanation is that defective *CYP2D6* alleles might consume less haem than normal alleles, so defective *CYP* allele carriers might be less prone to the overstimulation of haem synthesis. This last hypothesis would be supported if baseline levels of ALA and PBG were lower in defective *CYP2D6* allele carriers. However, we were unable to demonstrate that lower urinary ALA and PBG levels in defective *CYP2D6* allele carriers are directly associated with *CYP* genotype rather than acute attack history.

The strength of this study lies in the high genetic homogeneity in terms of a causal AIP mutation in *HMBS* gene, which eliminates biases due to differences in penetrance associated with various mutations. However, this study is not without limitations. The low allele frequencies of the *CYP* genetics variants and the limited number of patients studied make it necessary to confirm these findings in larger AIP populations. Further studies which include other *CYP* genes, as well as other candidate genes using next generation sequencing, might provide a more comprehensive overview of their possible modulating role in AIP.

Conclusions {#Sec9}
===========

AIP prevalence in the Spanish Region of Murcia is 17.7 cases/million inhabitants, almost 3 times higher than that estimated for the rest of Spain. The penetrance is 52%, similar to other founder mutation AIP populations. *CYP2D6\*4* and *\*5* alleles may be protective factors for acute attacks, and *CYP2D*6 may constitute a penetrance-modifying gene. Further studies are needed to confirm these findings, which would allow a further progress in AIP clinical risk profile assessment.

We highlight the relevance of knowing the prevalence and penetrance of AIP in a given population, especially in that ones with high prevalence of specific *HMBS* mutations, since published data vary among populations and mutations. These results will have a positive impact on AIP carriers genetic counselling and will allow a better planning of health care resources in our population. Furthermore, we emphasize the potential of applying *CYP* genotyping in AIP precision medicine, enabling personalized risk stratification of acute clinical manifestation based on individual *CYP* allelic profile and leading to predictive personalized medicine for each AIP carrier in the future.

AIP

:   Acute intermittent porphyria

ALA

:   Aminolevulinic acid

ALAS1

:   Aminolevulinic acid synthase 1

CYP

:   Cytochrome P450

HMBS

:   Hydroxymethylbilane synthase

LAIP

:   Latent acute intermittent porphyria

MAIP

:   Manifest acute intermittent porphyria

PBG

:   Porphobilinogen

We wish to acknowledge all the AIP patients and families for their generous participation, BIOBANC-MUR (IMIB-Arrixaca) for their contribution in the isolation and maintenance of DNA samples and PIER (Integral Plan of Rare Diseases in the Región de Murcia 2017-2020) for supporting research.

Funding {#FPar1}
=======

This research was partly founded by UCAM Universidad Católica de Murcia (grant PMAFI/09/14).

Availability of data and materials {#FPar2}
==================================

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

MBS and EGN conceived and planned the experiments; MJBM, VLG, MJS, LR, ATSR and EGN contributed to patient diagnosis. GG and EGN performed molecular characterization of AIP. MBS, EGN, JB, MJBM, VLG, MJS, LR and ATSR contributed to patient recruitment and the acquisition of clinical data; MBS, JB and RG contributed in sample collection (blood and urine); MBS performed genetic analysis in *CYP* genes; GG, MCM and PC supervised genetic analysis design and performance; CC performed statistical analysis; MBS and EGN contributed to the analysis of the results and wrote the manuscript; all authors provided critical feedback of analysis and helped shape the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate {#FPar3}
==========================================

This study was approved by the Research Ethics Board of the Hospital Clínico Universitario Virgen de la Arrixaca. Informed consent to participate was obtained from all patients.

Consent for publication {#FPar4}
=======================

Not applicable.

Competing interests {#FPar5}
===================

The authors declare that they have no competing interests.

Publisher's Note {#FPar6}
================

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


A high-yield double-purification proteomics strategy for the identification of SUMO sites.
The small ubiquitin-like modifier (SUMO) is a protein modifier that is post-translationally coupled to thousands of lysines in more than a thousand proteins. An understanding of which lysines are modified by SUMO is critical in unraveling its function as a master regulator of all nuclear processes, as well as its involvement in diseases such as cancer. Here we describe a protocol for the lysine-deficient (K0) method for efficient identification of SUMOylated lysines by mass spectrometry (MS). To our knowledge, the K0 method is the only currently available method that can routinely identify >1,000 SUMO sites in mammalian cells under standard growth conditions. The K0 strategy relies on introducing a His10-tagged SUMO wherein all lysines have been substituted to arginines. Lysine deficiency renders the SUMO immune to digestion by the endoproteinase Lys-C, which in turn allows for stringent and high-yield tandem purification through the His10 tag. In addition, the His10-tagged SUMO also contains a C-terminal Q87R mutation, which accommodates generation of SUMO-site peptides with a QQTGG mass remnant after digestion with trypsin. This remnant possesses a unique mass signature and readily generates diagnostic ions in the fragment ion scans, which increases SUMO-site identification confidence. The K0 method can be applied in any mammalian cell line or in any model system that allows for integration of the K0-SUMO construct. From the moment of cell lysis, the K0 method takes ∼7 d to perform.


LIMIT FROM 2000 RETURN 10881
BI-MONTHLY CANADA LIFE CONTRIBUTION 506.25
CANADA LIFE CONTRIBUTION UP TO DEC 31 2001 3590.30

WHAT ABOUT EMPLOYER PENSION PLAN CONTRIBUTIONS?
(2393.91)

CALL BMG

_________________________________________________________________
Get your FREE download of MSN Explorer at http://explorer.msn.com/intl.asp.


Q:

Which is the proper spelling: "disfunction" or "dysfunction"?

Is this word spelt dysfunction or disfunction?
Are there any correct spellings at all for this word?
The reason I asked is because I've always learned to spell it as "disfunction" until recently, when I realized I'm starting to see more of "dysfunction"

A:

Dysfunction is by far the more common according to Google Ngrams

A:

Hugo found a useful thread at Word Reference.
It points out that dys- is Greek in origin, and means "bad or difficult" and that function is Latin in origin and means "an activity that is natural to or the purpose of a person or thing". It goes on to say that the combination of Greek and Latin is somewhat odd, especially since dys-'s homophone dis- is Latin (but it does not mean the same as dys-).
The dis- prefix doesn't work for dysfunction because its meanings (as explained here) are not the same as dys-, so they would change the meaning of dysfunction.
The correct spelling is dysfunction. It would not surprise me, however, if disfunction became an accepted spelling, because it just seems more normal.


Q:

Determine transport method in SignalR Core?

In SignalR 2.0 the transport method can be determined by the transport parameter in the query string.
string transportMethod = queryString["transport"];

This doesn't seem to be the case in SignalR for ASP.NET Core.
The best I can do, it seems, is to use header information.
For WebSocket connections:
Connection = Upgrade
Upgrade = Websocket

And long polling:
Connection = Keep-Alive

And server-sent events:
Connection = Keep-Alive
Accept = text/event-stream

Is there a better/easier way to go about determining the transport method?

A:

You can get the transport type from the HubCallerContext like so
 Context.Items[ConnectionMetadataNames.Transport]


Time-dependent response theory and nonequilibrium free-energy relations.
The mathematics of time-dependent nonlinear response theory frequently leads to results which although formally exact, are not amenable to experimental application or even to use in computer simulations. Here we give a rigorous derivation of a tractable expression for the thermostatted nonlinear response of classical many body systems to time-dependent dissipative fields. The theory also allows for the concurrent parametric transformation of the system Hamiltonian. Our analysis shows once again the intimate relationship between nonequilibrium free-energy relations such as the Jarzynski equality and nonlinear response theory. We make a few remarks concerning the maximum entropy production approximation.


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Laurie Kilmartin

Aug 16 - Aug 18

Kilmartin began performing standup in 1987, doing contract comedy gigs in the Pacific Northwest. She has since performed stand-up on Conan, Jimmy Kimmel Live!, Comedy Central's Premium Blend, Showtime, the USO Tour Cuba, for troops in Iraq, Shorties Watchin' Shorties, Verdict with Dan Abrams, Red Eye w/ Greg Gutfeld, and The World Stands Up. She has been featured at comedy festivals in Aspen, Edinburgh, and Montreal.

She also appeared on VH1's Best Week Ever, the Today Show, White Boy.... more


1. Field of the Invention
This invention relates to manufacturing of a hollow plastic product and in particular to a method and apparatus for manufacturing a hollow plastic product having an irregular structure, such as a projection which projects outwardly from the outer surface of the product, or to a method of manufacturing a hollow plastic product having a change in composition. More specifically, the present invention relates to a blow molding technique for fabricating a hollow plastic product having a desired shape which cannot be manufactured by the conventional blow molding technology.
2. Description of the Prior Art
If it is desired to manufacture a hollow plastic product 2 having a projection 1 which projects outwardly from the outer surface of a main body 2' in accordance with the typical prior art technique, the main body 2' is first fabricated by blow molding and then the projection, which has been previously fabricated by any appropriate method, such as injection molding, is fixedly attached to the main body 2'. Such an approach is not advantageous because it requires a plurability of separate steps to carry out in manufacture and the connection between the main body 2' and the later-attached projection 1 is not reliable and low in strength. On the other hand, it is true that the projection 1 may be manufactured together with the main body 2' at the same time by blow molding; in this case, however, a significantly large flash is necessarily formed, and, thus it is expensive and time consuming to remove such a large flash, not to mention the production of significant waste of material.
Furthermore, it is sometimes desired that a hollow plastic product differs in composition depending on the location thereby providing different properties. For example, a hollow plastic tube is often desired to have a relatively hard section to be fixedly attached to a metal fitting or the like and another section which is relatively soft for providing an easy bending characteristic. In such a case, a typical prior art approach would be to first form different sections separately and then put them together to form a final product and such an approach is disadvantageous because such a multistep process would be time consuming and expensive and the integrity of the final product is relatively poor.


Diploma Jobs 2016

Many diploma holders are looking for latest Diploma jobs 2016. It is easy to the candidates to find out suitable jobs for you. The candidates will get diploma jobs according to their qualification. The candidate will get jobs based on their experience in particular field. The candidates who are completed their diploma will get suitable jobs the candidates can visit diploma notification to get more details about the qualification required for the posts. The recruitment board will evaluate your qualification by conducting various tests and all. So that the candidates have prepared well for the examination. It is important to get a job because it will decide their great future. All the diploma holders can select their job based on your skill and capacity. A lot of students are eagerly waiting for getting a good job.

Government Jobs for Diploma Holders 2016

The candidates who have completed their diploma can select suitable government jobs 2016. Many details are available in online the details include an application form, selection procedure, eligibility details and all, The candidates must have to notice that the last date for submitting the application form. Diploma Holders will get jobs in private and government company based on their eligibility. The candidates who have completed Engineering Diploma in recognized institution with minimum qualifying marks Computer Science (CS), Information Technology (IT), Electrical, Electronics and Communication, Mechanical, etc. are now searching for Jobs.

Diploma Holders Jobs 2016

Many Diploma holders are searching for latest diploma jobs 2016, and the candidates need to utilize their knowledge by working in the public and private sector companies. For the technician posts, the eligible candidates can apply in Bharat Sanchar Nigam Limited and Vidyut Vitran Nigam Limited. The candidates are still searching for the latest Diploma jobs in 2016. A lot of candidates are waiting for the good option to pick, and the candidates will get consolidated monthly pay scale according to organization norms.

Job vacancies for Diploma Holders

There are many job vacancies for the candidates who have completed their diploma in various fields. The candidates can apply for the post before the closing date. The candidates will get a job according to their qualification. As there are a lot of employment opportunities, it is easy to find Diploma Jobs 2016. The Candidates have to visit the notification and read the notification completely to get more information regarding educational qualification required for the post before applying for the posts.

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Diploma Holders can apply for the Recruitment of community development expert Field. There are many vacancies still available for diploma holders. AEE Civil and Office Assistant recruitment for Diploma holders in various fields. The diploma holders also apply for the post of a stenographer. For the posts, there will be a written examination. The candidates have to pass in the examination for the posts. In India, there are many vacancies for Diploma Holders for water supply and sewerage board.


Dynamic sympathetic regulation of left ventricular contractility studied in the isolated canine heart.
We investigated the dynamic sympathetic regulation of left ventricular end-systolic elastance (Ees) using an isolated canine ventricular preparation with functioning sympathetic nerves intact. We estimated the transfer function from both stellate ganglion stimulation to Ees and ganglion stimulation to heart rate (HR) for both left and right ganglia by means of the white noise approach and transformed those transfer functions into corresponding step responses. The HR response was much larger with right sympathetic stimulation than with left sympathetic stimulation (4.3 +/- 1.4 vs. 0.7 +/- 0.6 beats . min-1 . Hz-1, P < 0.01). In contrast, the Ees responses without pacing were not significantly different between left and right sympathetic stimulation (0.72 +/- 0.34 vs. 0.76 +/- 0. 42 mmHg . ml-1 . Hz-1). Fixed-rate pacing significantly decreased the Ees response to right sympathetic stimulation (0.53 +/- 0.43 mmHg . ml-1 . Hz-1, P < 0.01), but not to left sympathetic stimulation (0.67 +/- 0.32 mmHg . ml-1 . Hz-1, not significant). Although the mechanism by which the sympathetic nervous system regulates cardiac contractility is different depending on whether the left or right sympathetic nerves are activated, this difference does not affect the apparent response of Ees to dynamic sympathetic stimulation.


Q:

Why does it enter the same id in the database instead of entering one by one each?

Hy,
I want to ban a user when the admin clicks on "bannir" (this will collect the id of the user and put "banned" at 1 in the database)
to print the user informations I'm using a while loop, but when I try to collect the id of the user where in the html tag with the class "idUser", it is always sending the first id and I don't know why..
image of membership area

      #
      Prénom
      Nom
      Email
      Admin
      Banni
      Actions

    <?php while($userInfo = $req->fetch()){?>

      <?= $userInfo['id_user'] ?>
      <?= $userInfo['last_name'] ?>
      <?= $userInfo['first_name'] ?>
      <?= $userInfo['email'] ?>
      <?php if($userInfo['admin'] == 1){ ?>

        <?php } else { ?>

          <?php } ?>
      <?php if($userInfo['banned'] == 1){ ?>
        OUI
        <?php } else { ?>
        NON
          <?php } ?>
      Modifier | <?php if($userInfo["banned"] == 0){ ?> Bannir <?php }else{ ?> Débannir <?php } ?>

  <?php } ?>


Tottenham 17-18 Home Kit

The Nike Tottenham Hotspur 17-18 home kit introduces a clean look for the Swoosh's debut at the club. It was launched at midnight on July 1 due to contract obligations towards previous supplier Under Armour. This image shows the Nike Tottenham 2017-18 shirt.Made by Nike, the Tottenham 2017-2018 home jersey introduces clean and classy look that is based on the 2017 edition of the Nike Vapor Aeroswift template. The Tottenham 17-18 kit features a stripe detail around the back neck, while a blue stripe runs down the sides of it.To go with this understated and traditional design, the Tottenham 17-18 kit boasts a 1950s-inspired take on the Spurs badge with a shield around the original logo.Template-wise, the Tottenham 17-18 home shirt has a mesh knit detail on the sleeves as well as on the front and back (especially in the authentic version).The Nike Tottenham Hotspur 2017-2018 home kit was released at midnight on July 1 2017.Do you like the design of the first-ever Nike Tottenham Hotspur kit? Comment below, and check out all leaked and official 2017-18 Premier League kits in the Kit Overview


MHC class I recognition by NK receptors in the Ly49 family is strongly influenced by the beta 2-microglobulin subunit.
NK cell recognition of targets is strongly affected by MHC class I specific receptors. The recently published structure of the inhibitory receptor Ly49A in complex with H-2Dd revealed two distinct sites of interaction in the crystal. One of these involves the alpha1, alpha2, alpha3, and beta2-microglobulin (beta2m) domains of the MHC class I complex. The data from the structure, together with discrepancies in earlier studies using MHC class I tetramers, prompted us to study the role of the beta2m subunit in MHC class I-Ly49 interactions. Here we provide, to our knowledge, the first direct evidence that residues in the beta2m subunit affect binding of MHC class I molecules to Ly49 receptors. A change from murine beta2m to human beta2m in three different MHC class I molecules, H-2Db, H-2Kb, and H-2Dd, resulted in a loss of binding to the receptors Ly49A and Ly49C. Analysis of the amino acids involved in the binding of Ly49A to H-2Dd in the published crystal structure, and differing between the mouse and the human beta2m, suggests the cluster formed by residues Lys3, Thr4, Thr28, and Gln29, as a potentially important domain for the Ly49A-H-2Dd interaction. Another possibility is that the change of beta2m indirectly affects the conformation of distal parts of the MHC class I molecule, including the alpha1 and alpha2 domains of the heavy chain.


#!/usr/bin/env bash
# coding=utf-8
# Author: zhaigy@ucweb.com
# Data:   2012-09

. support/PUB.sh

for s in $NODES; do
  ssh $USER@$s " rm -f $HOME/.bash_profile.????????_??????; "
done


Jenkins\ Login=Jenkins prisijungimas
Access\ Denied=Prieiga atmesta


// Copyright (c) 2012 Google Inc. All rights reserved.
// Use of this source code is governed by a BSD-style license that can be
// found in the LICENSE file.

#include <stdio.h>

int hello2() {
  printf("Hello, two!\n");
  return 0;
}


Q:

How to properly search for data in a MySQL table

I have table in MySQL called movie like this
id    movie_name      movie_description
1   the godfather    The_godfather_description
2   the dark Knight  the_dark_knight_description
3   batman begins    batman_begins_description
4   Inception        Inception_description

When the query comes I am searching like this for each word in query
select movie_name,movie_description from movie where movie_name like(%is%) or movie_description like('%is%')
    select movie_name,movie_description from movie where movie_name like(%god%) or movie_description like('%god%')
   select movie_name,movie_description from movie where movie_name like(%father%) or movie_description like('%father%')

Can you tell me proper way to search database so that words like 'is' and 'the' get ignored.  I also want the most relevant articles on top.

A:

If I have got you correctly; you are searching for MySQL full Text search:
I want to explain you about Boolean Full Text Search; But I advise you to please go through Full Text Search using Query Expansion also.
Let's look at the example table:
mysql> select * from articles;
+----+-----------------------+------------------------------------------+
| id | title                 | body                                     |
+----+-----------------------+------------------------------------------+
|  1 | PostgreSQL Tutorial   | DBMS stands for DataBase ...             |
|  2 | How To Use MySQL Well | After you went through a ...             |
|  3 | Optimizing MySQL      | In this tutorial we will show ...        |
|  4 | 1001 MySQL Tricks     | 1. Never run mysqld as root. 2. ...      |
|  5 | MySQL vs. YourSQL     | In the following database comparison ... |
|  6 | MySQL Security        | When configured properly, MySQL ...      |
+----+-----------------------+------------------------------------------+

mysql> SELECT * FROM articles WHERE MATCH (title,body)
     AGAINST ('"database comparison"' IN BOOLEAN MODE);

+----+-------------------+------------------------------------------+
| id | title             | body                                     |
+----+-------------------+------------------------------------------+
|  5 | MySQL vs. YourSQL | In the following database comparison ... |
+----+-------------------+------------------------------------------+

Order matters, when the words are quoted:
mysql> SELECT * FROM articles WHERE MATCH (title,body)
     AGAINST ('"comparison database"' IN BOOLEAN MODE);

Empty set (0.01 sec)

When we remove the quotes, it will search for rows, containing words "database" or "comparison":
mysql> SELECT * FROM articles WHERE MATCH (title,body)
     AGAINST ('database comparison' IN BOOLEAN MODE);

+----+---------------------+------------------------------------------+
| id | title               | body                                     |
+----+---------------------+------------------------------------------+
|  1 | PostgreSQL Tutorial | DBMS stands for DataBase ...             |
|  5 | MySQL vs. YourSQL   | In the following database comparison ... |
+----+---------------------+------------------------------------------+

Order doesn't matter now:
mysql> SELECT * FROM articles WHERE MATCH (title,body)
     AGAINST ('comparison database' IN BOOLEAN MODE);

+----+---------------------+------------------------------------------+
| id | title               | body                                     |
+----+---------------------+------------------------------------------+
|  1 | PostgreSQL Tutorial | DBMS stands for DataBase ...             |
|  5 | MySQL vs. YourSQL   | In the following database comparison ... |
+----+---------------------+------------------------------------------+

If we want to get rows, containing either word "PostgreSQL" or phrase "database comparison", we should use this request:
mysql> SELECT * FROM articles WHERE MATCH (title,body)
     AGAINST ('PostgreSQL "database comparison"' IN BOOLEAN MODE);

+----+---------------------+------------------------------------------+
| id | title               | body                                     |
+----+---------------------+------------------------------------------+
|  1 | PostgreSQL Tutorial | DBMS stands for DataBase ...             |
|  5 | MySQL vs. YourSQL   | In the following database comparison ... |
+----+---------------------+------------------------------------------+

fiddle
Make sure, that the words, you are searching for, are not in the list of stopwords, that are ignored.
(and obviously words like 'is','the' are stopwords and those are ignored)
I have explained here only about Boolean Full Text Search; But I advise you to please go through Full Text Search using Query Expansion also.


The physician assistant profession and military veterans.
The physician assistant (PA) profession originated to train former medics and corpsmen for a new civilian health care career. However, baccalaureate degree prerequisites to training present barriers to discharged personnel seeking to enter this profession. A survey was administered (2006-2007) to all MEDEX Northwest PA program graduates who had entered with military experience. The survey addressed attitudes toward the profession, PA education, and practice and how military experience influenced their education and careers. The response rate was 46.4%, spanning all branches of the military. Respondents reported military experience positively impacting ability to handle stress and work in health care teams and that patients and colleagues viewed their military background positively. Most (75.5%) respondents did not hold a bachelor's degree at matriculation. Veterans bring substantial health care training to the PA profession. However, program prerequisites increasingly present barriers to entry. Veterans' contributions to health care and the consequences of losing this resource are discussed.


Donald Trump’s victory over Clinton isn’t just impressive because it showcased a skeletal campaign outfit defeating a team that outsized it in terms of experience and infrastructure. What is truly jaw-dropping is how a campaign operation such as Trump’s overcame the odds against a rival organization that out-fundraised it by shocking proportions.

How much did Hillary Clinton actually raise during this campaign? Her campaign raked in just shy of 498 million dollars. However, other outside outfits such as her Super PAC (Priorities USA ACTION) and other aligned groups raised a little over $189 million. A huge sum when put together. Priorities USA raised more money than any Super PAC ever – the vast majority of the cash raised came from seven figure donations.

Donald Trump was not only up against an operation that executed impeccable fundraising prowess. He also lacked the resources to fundraise within the same ballpark. Trump’s team only raised $247 million. He took in $380 million less than the Democratic nominee. His 2016 war chest came nowhere close to Romney’s $450 million haul – a candidate who ran for president four years ago.

This disparity in fundraising trickled down to campaign expenditures. Altogether, Trump spent about five dollars for each vote. Clinton spent about $10.

The point being is that Trump did more with less. Clinton paid twice as much for her voters and still came short in the Electoral College. Trump lost the money war but still ended Election Day victorious.

Why did liberal mega-donors get such a terrible return on investment with their candidate? They are meeting to understand why and how to not repeat their same mistakes.

Rich liberals across the country (George Soros included) are meeting for a three-day, private conference in Washington to discuss strategy moving forward. Union leaders, liberal billionaires, Nancy Pelosi, Elizabeth Warren, and Keith Ellison are some of the bigwigs attending. The event is hosted by the Democracy Alliance donor club. An outline of their schedule was obtained by POLITICO.

The topic of discussion will be how best to utilize their political donations moving forward. The focus will not just be on the next national election, but how to be more successful in races at the local and state level – an area Democrats have performed terribly in.

George Soros and others in the group believe the best way to halt Trump’s agenda and protect Obamacare is to win more state legislatures and gubernatorial seats. Rep. Keith Ellison (D) is a part of the talks and is pushing the group to engage working class voters – the voting block that went in droves for Trump.

Ellison, Congress’ first Muslim representative, is also being positioned as the strongest in line to become the DNC chairman. If he succeeds Donna Brazile, it will be a sharp left turn for an already far-left Democratic Party. He's been in talks with Party leaders and their wealthy donors.

Despite their losses, be prepared for the liberal elite to keep spending millions to promote their progressive agenda on the country. They should keep in mind, however, that Trump has proven himself knowledgeable on how to use his own resources much more efficiently.


**Core tip:** In this study, we summarized and analyzed the mRNA expression data of TCGA-COAD and obtained the expression features of AMPD2 in cancer tissues and adjacent tissues in patients with colorectal cancer. We constructed cell samples that were not subjected to high-throughput sequencing of RNA-seq, and although the results were closer to clinical reality, in the case of verification experiments, the probability of false negatives was increased. In addition, our cytology validation experiments need to be carried out in more detail, and our colorectal cancer cohort sample was only 201 cases. We expect to add cytological evidence and a large sample of prospective prognostic cohorts in subsequent studies.

INTRODUCTION
============

Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related deaths worldwide, and remains a major health threat in China\[[@B1],[@B2]\]. CRC patients at an advanced stage often have a poor prognosis due to abnormal tumor metabolism and recurrence. Therefore, it is of clinical significance to identify novel biomarkers that control the severity of CRC as well as biomarkers for the prediction of prognosis\[[@B3]\].

Recently, there has been increased interest in studying the metabolic reprogramming in cancer in the hope of developing metabolism-based anticancer therapy\[[@B4]\]. Accordingly, various studies aimed at identifying the metabolic index in blood, body fluids, carcinoma tissue, and gut microbiota have been performed to establish a complete metabolic atlas and explain the possible mechanisms by which these abnormalities are present in gastrocolorectal tumors\[[@B5]\]. In several studies, it has been shown that in addition to primary metabolism, many specific secondary metabolites have an important impact on the development and progression of CRC, thereby suggesting that specific metabolic markers can be used as biomarkers for metabolic abnormalities\[[@B6]\]. These findings suggested that AMPD2 may act as a metabolic tissue enzyme, which is closely related to the type of CRC, and may play an important role in CRC.

AMPD2 is an important enzyme involved in adenosine monophosphate metabolism and plays an important biological function in adenylate metabolism in smooth muscle tissue\[[@B7]\]. In previous studies, it was shown that AMPD2 affected the nephrotic syndrome and hypercholesterolemia\[[@B8]\]. In addition, it has been demonstrated that AMPD2 dramatically affected physiological and pathological processes\[[@B9]\]. Despite recent efforts to understand the activities of AMPD2 in the biological mechanism of cancer, little is known about the role of AMPD2 in CRC and it remains to be elucidated whether AMPD2 expression correlates with any of the clinicopathological features of CRC\[[@B10]\].

In this study, we combined TCGA-COAD data set analyses of both tumor and normal samples to investigate the significance of AMPD2 in CRC. Therefore, *in vitro* studies were performed to investigate how AMPD2 may affect the CRC-associated regulatory pathway. A total of 201 samples were collected using tissue microarray validation cohort studies to investigate whether AMPD2 could be used as a biomarker for poor CRC prognosis.

MATERIALS AND METHODS
=====================

Data collection and preprocessing analysis
------------------------------------------

In this study, the TCGA dataset was used for analysis of gene expression in CRC tissues and normal tissues. TCGA-COAD RNA-seq data and related clinical data of cancer tissues (478), and normal tissues (41) were downloaded from <https://>[xenabrowser.net/](xenabrowser.net/)\[[@B11]\]. We continued to match and screen the organization label, and obtained 40 pairs of cancer tissues and paired adjacent normal tissues. The data were updated to November 2018. To assess the prospective functions of the genes influenced by AMPD2, we selected a total of 50 TCGA-COAD specimens with the highest expression of AMPD2 and 50 TCGA-COAD specimens with the lowest expression of AMPD2, and follow-up biologic function prediction analysis was performed in the abnormally high expression and low expression groups. Gene Ontology (GO)\[[@B12]\] and Kyoto Encyclopedia of Genes and Genomes (KEGG)\[[@B13]\] analyses were employed using R software (version 3.6) Bioconductor module. The datasets GSE97023 were downloaded from the Gene Expression Omnibus (GEO) (<https://www.ncbi.nlm.nih.gov/geo/>)\[[@B14]\], to analyze RNA-seq baseline data for CRC cell lines.

Cell lines and cell culture, and AMPD2 overexpression and knockdown
-------------------------------------------------------------------

Human CRC cell lines SW480 and Co115 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's Modified Eagle's Medium (Biowest, France) with 10% fetal bovine serum (Gibco, United States) and 1% penicillin-streptomycin antibiotics (Gibco, United States). Cells were cultured in an incubator at 37°C with 5% CO~2~. For AMPD2 overexpression, cells were transfected with AMPD2 overexpression plasmid or blank plasmid purchased from Gene Pharma (Shanghai, China). For AMPD2 knockdown, an AMPD2 specific siRNA (siAMPD2) and a negative control siRNA (siNEG) were chemically synthesized by Gene Pharma (Shanghai, China). Transfection of cells with siRNA was performed using Lipofectamine 2000 (Invitrogen, ThermoFisher Scientific, United States) according to the manufacturer's guidelines.

Western blot analysis
---------------------

Cells were harvested and lysed in RIPA buffer, containing proteinase and phosphatase inhibitors. The concentration of proteins in cell lysates was determined by a BCA kit (Applygen Technologies, China) following the manufacturer's instructions. Equal amounts of cell lysates (20 μg protein/lane) were electrophoresed on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked, and then probed overnight with appropriate primary antibodies (AMPD2, 1:1000; Notch3, 1:500; GAPDH, 1:8000) in blocking buffer at 4°C. Bound antibodies were detected with Beyotime horseradish (HRP)-conjugated goat anti-rabbit IgG or anti-mouse IgG (at 1:10000 dilution; Genetech, China) and visualized using a Bio-Rad ChemiDoc XRS imaging system.

Tissue microarray
-----------------

A total of 201 CRC patients who underwent CRC surgery between January and August 2014 in the Oncology Department, Affiliated Hospital of Jiangnan University, Wuxi, China, were enrolled in the present study. The patients were followed-up until December 2018. Based on the patient\'s medical records, formalin-fixed paraffin-embedded (FFPE) tissue specimens from these patients were collected. Clinical and pathological parameters, including patient age, gender, tumor size, histological grade, primary tumor (pT), lymph node (pN) metastasis, pathological staging, neurological invasion, lymphatic invasion, and vascular invasion were analyzed. Patient data and tissue samples collected during our study were approved by the Ethics Committee of Affiliated Hospital of the Jiangnan University (Jiangnan, China). After collecting the paraffin blocks, we fabricated tissue microarrays (TMA) using a Tissue microarray sampler (Quick-Ray; UNITMA, Korea). From each colorectal adenocarcinoma tissue, two representative tumor cores with a diameter of 1.5 mm were obtained. Hematoxylin and eosin (H&E) staining was performed on each tissue to confirm tumor tissue integrity and cell morphology. Cases with insufficient cancer tissue or cases with no adenocarcinoma in the core were not included in the study. To ensure the stability of subsequent TMA studies, two areas were sampled in each tumor tissue and normal epithelial tissue.

Immunohistochemical staining
----------------------------

Immunohistochemistry was performed using 4 µm sections from FFPE tissue specimens of CRC patients. After incubation in xylene three times for five minutes each time, and subsequent passages in gradient alcohol, the sections were immersed in distilled water for 5 min. Next, an antigen retrieval procedure was performed using a sodium citrate solution for 20 min in boiling water. Sections were incubated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase. Subsequently, the sections were incubated overnight at 4°C with a primary polyclonal antibody directed against AMPD2 (1:200, ProteinTech, China) and Notch3 (1:200, ProteinTech, China). The sections were washed, incubated with the amplification agent and polymerase (reagent A, GTVisionTM III Kit supply, China), then stained with 3,3'-diaminobenzidine (reagent B and C, GTVisionTM III Kit supply, Shanghai, China). Sections were counterstained using hematoxylin. In each section, the staining was scored according to the percentage of positively-stained cells in the entire section (no positive staining or ≤ 5% = 0; 6%-25% positive = 1; 26%-50% positive = 2; 51%-75% positive = 3; and 76%-100% positive = 4). The scoring intensity was determined as previously described: no staining = 0, weak staining = 1, moderate staining = 2, strong staining = 3\[[@B15]\].

Statistical analysis
--------------------

Cut-off values for AMPD2 and Notch3 were obtained from the receiver operating characteristic curve (ROC) and the area under curve (AUC). Pearson's Chi-square tests or Fisher's exact tests were employed to analyze the association between AMPD2 expression, clinicopathological parameters, and Notch3. Moreover, Kaplan-Meier survival analysis was used to determine the probability of overall survival (OS). Data were analyzed using log-rank tests. Univariate and multivariate survival analyses were performed using the Cox proportional hazard model and OS. Statistical analyses were performed using SPSS for Windows 22.0 software (IBM). *P* \< 0.05 was considered statistically significant.

RESULTS
=======

AMPD2 is commonly overexpressed in TCGA CRC tissues
---------------------------------------------------

In this study, we evaluated AMPD2 expression in CRC and normal colorectal tissues, and changes in transcriptional levels of AMPD2 were first validated using microarray values in all tumor tissue and normal tissue from the TCGA-COAD data sets. The results showed that AMPD2 mRNA was significantly increased in CRC tissues in the TCGA-COAD data set (*P* = 0.0238; Figure [1A](#F1){ref-type="fig"}). Moreover, we demonstrated that an increase in AMPD2 mRNA abundance was observed in paired cancerous tissue when compared with paired adjacent normal tissue from the TCGA data set (Figure [1B](#F1){ref-type="fig"}). In addition, we used the TCGA-COAD data set to correlate mRNA expression with AMPD2. A total of 50 specimens with abnormally-high expression of AMPD2 and 50 specimens of abnormally-low expression of AMPD2 were selected, and differential gene expression was analyzed. According to the high and low expression of AMPD2, a heat map was drawn as shown in Figure [1C](#F1){ref-type="fig"}.

![The expression level of AMPD2 in the TCGA-COAD data set. A: The expression level of AMPD2 in cancer tissues and paracancerous tissues was determined by two independent samples *t* test, *P* \< 0.05 was considered significant; B: In paired samples of cancer tissue and adjacent normal tissues, the AMPD2 expression level was determined by paired t-test, and *P* \< 0.05 was considered significant; C: AMPD2-based high expression subgroup (50 cases) and low expression subgroup (50 cases) in cancer tissues. A heat map of the difference in gene expression was produced.](WJCC-8-3197-g001){#F1}

Functional implication of AMPD2 associated with the Notch pathway in CRC in vitro
---------------------------------------------------------------------------------

To further investigate the biological function of the differential expression of transcription levels caused by high expression of AMPD2, we performed GO and KEGG enrichment analysis of the genes that were up-regulated in the AMPD2 overexpression group (Figure [2A and B](#F2){ref-type="fig"}). When compared to the AMPD2 low expression group, the AMPD2 overexpression group showed a positive regulation of neural precursor cell proliferation. In addition, KEGG analysis showed that the Notch pathway was strongly correlated with AMPD2 expression, and the expression of Notch3 and JAG2 mRNA was positively associated with AMPD2 in CRC tissues. These results suggested that high expression of the Notch signaling pathway in CRC patients with AMPD2 up-regulation affected the progression of CRC.

![Functional enrichment analysis and western blot. A and B: Based on AMPD2, a high expression subgroup (50 cases) and a low expression subgroup (50 cases) in cancer tissues were identified, and functional enrichment analysis of gene expression differences was performed using GO and KEGG maps; C: The expression of AMPD2 in various colorectal cancer cell lines was analyzed according to the GEO-GSE97023 data set; D and E: The Western blot results of up-regulation and down-regulation of AMPD2 in SW480 and Co115 cell lines, immunoblotting (D) and histogram (E).](WJCC-8-3197-g002){#F2}

Figure [2C](#F2){ref-type="fig"} shows AMPD2 (gene probe: TC01000950.hg.1) baseline mRNA expression values in various CRC cell lines. We selected SW480 and Co115 cell lines for subsequent up-regulation and silencing studies of cell biology. Protein expression levels were determined. In brief, AMPD2 and Notch3 protein expression was evaluated in SW480 and Co115 cells. Both AMPD2 and Notch3 were up-regulated at the protein level (Figure [2D and E](#F2){ref-type="fig"}) in SW480 and Co115 cells with AMPD2-Lent when SW480 and Co115 cells were compared with the AMPD2 negative control. Similarly, both AMPD2 and Notch3 were down-regulated at the protein level (Figure [2D and E](#F2){ref-type="fig"}) in SW480 and Co115 cells treated with siAMPD2 when compared to SW480 and Co115 cells that were transfected with the negative control.

Correlation between AMPD2 expression and clinicopathological features in CRC
----------------------------------------------------------------------------

We analyzed the correlation between high expression of AMPD2 and Notch3 and clinicopathological parameters in CRC patients. Clinicopathological and demographic characteristics are presented in Table [1](#T1){ref-type="table"}. Immunohistochemical analysis of AMPD2 and Notch3 expression in 201 CRC patients revealed that AMPD2 and Notch3 were predominantly expressed in the cytoplasm of carcinoma cells (Figure [3A](#F3){ref-type="fig"}). To assess the classification of AMPD2 and Notch3 protein expression, we used the immunohistochemical semi-quantitative score and predicted the ROC curve for survival by AMPD2 and Notch3 expression levels, and calculated the cut-off value in conjunction with the results of the Youden index. Thus, patients were categorized as high expression of AMPD2 if their immunohistochemical scores were \> 0.50. As shown in Table [1](#T1){ref-type="table"}, overexpression of AMPD2 significantly correlated with advanced depth of the tumor (*P* = 0.022) and poor differentiation (*P* = 0.037). As shown in Table [2](#T2){ref-type="table"}, univariate Cox analysis for OS indicated a correlation with higher TNM stage (HR = 3.198, 95%CI: 1.915-5.343, *P* \< 0.001) and increased expression of AMPD2 (HR = 1.769, 95%CI: 1.094-2.862, *P* = 0.020). Furthermore, multivariate regression analysis indicated that a higher TNM stage was associated with a relative risk of death of 3.727 (95%CI: 2.206-6.296, *P* \< 0.001) when compared with that of Stage I-II. In addition, overexpression of AMPD2 was associated with a relative risk of death of 2.133 (95%CI: 1.295-3.513, *P* = 0.003). Patients with colorectal adenocarcinomas who belonged to the AMPD2 high expression group (*n* = 61) had a significantly lower cumulative survival rate when compared to patients in the AMPD2 low expression group (*n* = 140) (log-rank *P* = 0.0181). Patients with CRC who had high expression of Notch3 (*n* = 70) had a significantly lower cumulative survival rate when compared to those with low expression of Notch3 (*n* = 131) (log-rank *P* = 0.465). Subgroup survival analysis revealed that patients who showed high expression of both AMPD2 and Notch3 had the worst prognosis when compared with the other three subgroups. Patients with low expression of AMPD2 and low expression of Notch3 showed the highest survival when compared to the other subgroups (*P* \< 0.001, Figure [3B](#F3){ref-type="fig"}).

######

Correlations between clinicopathological characteristics and both AMPD2 and Notch3 expression in 201 colorectal cancer patients

  **Parameters**      **Total (*n* = 201)**   **AMPD2 expression**                    **Chi-square value**   ***P* value**
  ------------------- ----------------------- --------------------------------------- ---------------------- --------------- --
  Gender              0.664                   0.415
  Male                114                     32                                      82
  Female              87                      20                                      67
  Age (yr)            0.004                   0.953
  \< 60               78                      20                                      58
  ≥ 60                123                     32                                      91
  Location            3.045                   0.081
  Colon               80                      26                                      54
  Rectum              121                     26                                      95
  Vascular invasion   0.502                   0.479
  Negative            165                     41                                      124
  Positive            36                      11                                      25
  Neural invasion     0.780                   0.337
  Negative            170                     42                                      128
  Positive            31                      10                                      21
  Depth of tumor      5.270                   0.022[a](#T1FN1){ref-type="table-fn"}
  T1/T2               60                      9                                       51
  T3/T4               141                     43                                      98
  Lymph metastasis    0.048                   0.826
  N0                  94                      25                                      69
  N1/N2               107                     27                                      80
  Differentiation     3.887                   0.037[a](#T1FN1){ref-type="table-fn"}
  Well/mod            184                     44                                      140
  Poor                17                      8                                       9
  TNM stage           0.487                   0.485
  I-II                96                      27                                      69
  III-IV              105                     25                                      80
  Notch3              0.389                   0.553
  High                140                     38                                      102
  Low                 61                      14                                      47

*P* \< 0.05 was considered statistically significant.

######

Cox proportional model predicts the relationship between AMPD2, Notch3 and clinicopathological parameters and survival

  **Clinicopathological** **Parameters**   **Univariate**        **Multivariate**
  ---------------------------------------- --------------------- ------------------------------------------ --------------------- -----------------------------------------
  Gender (female/male)                     1.004 (0.636-1.585)   0.986                                      0.973 (0.610-1.552)   0.909
  Age (≥ 60/\< 60)                         1.060 (0.664-1.693)   0.806                                      1.066 (0.661-1.721)   0.793
  Location (rectum/colon)                  0.889 (0.561-1.409)   0.616                                      0.888 (0.553-1.428)   0.625
  Differentiation (poor, moderate/well)    0.496 (0.249-0.988)   0.041                                      0.727 (0.450-1.173)   0.191
  TNM stage (III-IV/I-II)                  3.198 (1.915-5.343)   \< 0.001[a](#T2FN1){ref-type="table-fn"}   3.727 (2.206-6.296)   \<0.001[a](#T2FN1){ref-type="table-fn"}
  AMPD2 (high/low)                         1.769 (1.094-2.862)   0.020[a](#T2FN1){ref-type="table-fn"}      2.133 (1.295-3.513)   0.003[a](#T2FN1){ref-type="table-fn"}
  Notch3 (high/low)                        1.237 (0.747-2.048)   0.408                                      1.119 (0.668-1.874)   0.669

*P* \< 0.05 was considered statistically significant. HR: Hazard ratio; CI: Confidence interval.

![Immunohistochemical and Kaplan-Meier curves. A: Tissue microarrays of hematoxylin and eosin stained sections. Representative immunohistochemical analysis of AMPD2 levels in colorectal tumor tissue specimens from cancer patients and in adjacent cancer-associated fibroblasts. Immunohistochemical results of Notch3 levels in the same tissues/cells. Photomicrographs of the sections (50 × and 200 × magnification); B: Kaplan-Meier curves showing the impact of AMPD2 expression on overall survival (OS) of 201 colorectal cancer patients (*P* = 0.0181). Kaplan-Meier curves showing the impact of Notch3 expression on the OS of 201 colorectal cancer patients (*P* = 0.465). Kaplan-Meier curves showing the impact of AMPD2 and Notch3 subgroup expression on the OS of 201 colorectal cancer patients (*P* \< 0.001).](WJCC-8-3197-g003){#F3}

DISCUSSION
==========

Metabolic intermediate and related metabolites are widely present in cells of myometrial and glandular tissues\[[@B16]\]. Their associated metabolic pathways have been reported to be central to cancer growth and metastasis\[[@B17]\]. Thus, upregulation of AMPD2 in the tumor may explain the metabolic shift in adenosine phosphate synthesis in cancer. This evidence from different studies robustly supports that AMPD2 might be the fundamental gene in tumor transformation, resulting in metabolic adaptation in cancer cells\[[@B18]\]. Based on these findings, we suggested that AMPD2 may be a tumor metabolic marker that can be used for the analysis of tumor progression and the prediction of patient prognosis.

In this study, we summarized and analyzed the mRNA expression data of TCGA-COAD, and obtained the expression characteristics of AMPD2 in cancer tissues and adjacent tissues in patients with CRC. A gene function prediction analysis of the differences between high and low AMPD2 expression groups was performed, and the results were enriched in the Notch signaling pathway. Based on these findings, cytological mechanism validation and clinical prognosis-related survival analysis were employed. Our data showed that AMPD2 resulted in CRC progression and poor prognosis in colorectal and CRC tissues. However, our analysis based on gene function enrichment was based on TCGA public database analysis. We constructed cell samples that were not subjected to high-throughput sequencing of RNA-seq. The results were closer to clinical reality; however, when verifying the data, the probability of false negatives was increased. In addition, our cytology validation experiments needed to be carried out in detail, and our CRC cohort sample only included 201 cases. We expect to add cytological evidence and increase the sample size of prospective prognostic cohorts in subsequent studies.

In the present work, we revealed for the first time that overexpression of AMPD2 can promote CRC progression by augmenting tumor proliferation and halting apoptosis, and may serve as a poor prognostic marker for CRC patients. In addition, our data provided evidence that the Notch signaling pathway can affect proline metabolism *via* AMPD2 in CRC. Taken together, these findings, and our previous report, highlight the potential therapeutic value of targeting AMPD2 as a modulator of proline metabolism in CRC.

ARTICLE HIGHLIGHTS
==================

Research background
-------------------

As an important gastrointestinal tumor, colorectal cancer (CRC) is often accompanied by abnormal energy metabolism in the case of malignant progression and poor prognosis.

Research motivation
-------------------

As the abnormal energy metabolism in tumor cells has received considerable attention, we have combined the tissue type and biological characteristics of CRC. With AMPD2 as the entry point for research, a variety of scientific methods can be integrated for research.

Research objectives
-------------------

Clarify the role of AMPD2 in CRC and study the pathway and prognostic value of its role.

Research methods
----------------

RNAseq data analysis of the TCGA-COAD data set, GO and KEGG analysis. Western blot was used to detect the expression of Notch3-related pathway proteins, as well as clinical pathological samples combined with patient prognosis analysis.

Research results
----------------

AMPD2 is commonly overexpressed in TCGA CRC tissues, and the function of AMPD2 is associated with the Notch signaling pathway in CRC *in vitro*. In the CRC cohort, as indicated by tissue microarray analysis, high expression of AMPD2 protein was correlated with advanced depth of tumor and poor differentiation.

Research conclusions
--------------------

The mRNA and protein levels of AMPD2 are significantly highly expressed in CRC tissues. The high expression of AMPD2 has a positive relationship with activation of the Notch3 signaling pathway. In patients with CRC, those with high expression of AMPD2 have a poor prognosis.

Research perspectives
---------------------

In the future, we plan to further study the biological mechanism of AMPD2 stimulation of abnormal lipid metabolism in CRC cells.

ACKNOWLEDGEMENTS
================

The authors thank Yin ZG and Wang ZW of the Department of Pathology, Affiliated Hospital of Jiangnan University for their help in this study.

Institutional review board statement: This study was approved by the Ethics Committee of Affiliated Hospital of Jiangnan University and was performed in accordance with the ethical standards of the Declaration of Helsinki and its later amendments.

Clinical trial registration statement: This registration policy applies to prospective, randomized, controlled trials only.

Informed consent statement: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.

Conflict-of-interest statement: The authors declare that they have no competing interests.

Manuscript source: Unsolicited manuscript

Peer-review started: March 24, 2020

First decision: April 24, 2020

Article in press: July 15, 2020

Specialty type: Medicine, research and experimental

Country/Territory of origin: China

Peer-review report's scientific quality classification

Grade A (Excellent): 0

Grade B (Very good): B

Grade C (Good): C, C

Grade D (Fair): 0

Grade E (Poor): 0

P-Reviewer: Mattioli AV, Kopljar M, Sipos F S-Editor: Yan JP L-Editor: Webster JR E-Editor: Xing YX

[^1]: Author contributions: Gao QZ and Qin Y designed the research; Gao QZ performed the research. Qin Y and Jin JQ reviewed the surgical pathology; Fei BJ and Han WF analyzed the data; Gao QZ and Gao X wrote the paper; all authors read and approved the final manuscript.

    Supported by the Wuxi Young Medical Talents, Jiangsu Province, China, No. QNRC063.

    Corresponding author: Xiang Gao, MD, Academic Research, Department of Oncology, Affiliated Hospital of Jiangnan University, No. 200 Huihe Road, Wuxi 214062, Jiangsu Province, China. <13606189128@139.com>