Long noncoding RNA PTPRG-AS1 acts as a microRNA-194-3p sponge to regulate radiosensitivity and metastasis of nasopharyngeal carcinoma cells via PRC1

Protein regulator of cytokinesis 1 (PRC1) has been reported in correlation with various malignancies. Functionality of PRC1 in nasopharyngeal carcinoma (NPC) was investigated, in perspective of long noncoding RNA (lncRNA) regulatory circuitry. Aberrant expressed messenger RNA and lncRNA were screened out from the Gene Expression Omnibus microarray database. NPC cell line CNE-2 was adopted for in vitro study and transfected with mimic or short hairpin RNA of miR-194-3p and PTPRG-AS1. The radioactive sensitivity, cell viability, migration, invasion, and apoptosis were detected. PTPRG-AS1 and PRC1 were upregulated in NPC, whereas miR-194-3p was downregulated. PTPRG-AS1 was found to specifically bind to miR-194-3p as a competing endogenous RNA and miR-194-3p targets and negatively regulates PRC1. Overexpressed miR-194-3p or silenced PTPRG-AS1 resulted in enhanced sensitivity to radiotherapy and cell apoptosis along with suppressed cell migration, invasion and proliferation in NPC. Furthermore, impaired tumor formation was also caused by miR-194-3p overexpression or PTPRG-AS1 suppression through xenograft tumor in nude mice. In our study, PTPRG-AS1/miR-194-3p/PRC1 regulatory circuitry was revealed in NPC, the mechanism of which can be of clinical significance for treatment of NPC.     

LncRNA PCED1B-AS1 activates the proliferation and restricts the apoptosis of glioma through cooperating with miR-194-5p/PCED1B axis

Glioma with poor prognosis is accepted as a lethal, malignant intracranial tumor among central nervous system diseases. It has been frequently exhibited that long noncoding RNAs (lncRNAs) exert indispensable functions in glioma through regulating gene expression through various molecular mechanisms. To unveil a novel lncRNA functioning in glioma, we browsed the cancer genome atlas dataset and chose the lncRNA PC-esterase domain containing 1B antisense RNA 1 (PCED1B-AS1) for further investigations. Loss-of-function experiments depicted that the proliferation ability was restrained and apoptosis ability was induced in glioma cells by PCED1B-AS1 silencing and this phenomenon was also observed when PCED1B was knocked down. In view of the position of PCED1B-AS1 in the cytoplasm, we produced the Venn diagram and discovered one shared microRNA of PCED1B-AS1 and PCED1B. The competing endogenous RNA network formed by PCED1B-AS1, miR-194-5p, and PCED1B was attested by mechanism assays. Rescue experiments reconfirmed that miR-194-5p suppression or PCED1B overexpression neutralized the obstructive impacts of PCED1B-AS1 silence on proliferation and the promoting effects of PCED1B-AS1 silence on apoptosis. The modulation mechanism of the PCED1B-AS1/miR-194-5p/PCED1B axis in glioma was investigated and affirmed, which supports researchers with a new insight into the therapy of patients with glioma.     

The Arg194Trp polymorphism in the XRCC1 gene and cancer risk in Chinese Mainland population: a meta-analysis

The Arg194Trp polymorphism in the X-ray repair cross-complementing group 1 (XRCC1) gene has been proved to be in association with cancer risk in Chinese Mainland population, but a large number of studies have reported inconclusive results. A more comprehensive and precise estimation of the relationship is needed to clear the way towards future studies. Thus, we performed a meta-analysis to analysis these associations. A total of 34 case-control studies in 34 articles were included in this meta-analysis. The results showed that the 194Trp/Trp homozygote had a 31% increased risk of cancer than 194Trp/Arg and 194Arg/Arg genotypes, OR was 1.31 and 95%CI was 1.13 to 1.53. In the subgroup analysis by cancer sites, the Arg194Trp polymorphism was associated with increased risks of lung cancer (OR = 1.27 and 95%CI: 1.07-1.50 for Trp/Trp versus Arg/Arg + Arg/Trp) and esophageal cancer (OR = 1.68 and 95%CI: 1.33-2.13 for Trp/Trp versus Arg/Arg + Arg/Trp). This meta-analysis suggested that the Arg194Trp polymorphism of the XRCC1 gene is a cancer susceptible factor among Chinese Mainland population. More intensive and deeper studies are needed to further reveal the mechanism between Arg194Trp polymorphisms of XRCC1 gene and cancer risks in Chinese Mainland population.     

Characterization of functional heme domains from soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.     

Sequence of the viral replicase gene from foot-and-mouth disease virus C1-Santa Pau (C-S8)

The nucleotide sequence of the region including the viral replicase gene, the carboxy terminus of protein P18, and the 3'-extracistronic region of foot-and-mouth disease virus (FMDV) type C1-Santa Pau (C-S8) has been determined from previously cloned cDNA fragments [Villanueva et al., Gene 23 (1983) 185-194]. The comparison with the corresponding gene segments of FMDV of serotypes A or O shows base substitutions in 7.2-8.6% of residues in the replicase gene with no insertions or deletions. This is about fourfold lower variation than found for the region encoding capsid protein VP1 of the corresponding viruses. Intermediate variability (substitution at 16.1-23.6% positions) exists in the 3'-extracistronic region, including point mutations, insertions and deletions. The predicted amino acid sequence of the replicase gene indicates that 75.5-82.6% of mutations are silent and that 93.4% of amino acids are conserved in the four FMDV replicases. The frequency of certain types of silent mutations and of rare codon usage is significantly lower for the replicase gene than for the protein VP1 coding region.     

Crystal structures of the reaction intermediate and its homologue of an extradiol-cleaving catecholic dioxygenase

BphC derived from Pseudomonas sp. strain KKS102 is an extradiol-cleaving catecholic dioxygenase. This enzyme contains a non-heme iron atom and plays an important role in degrading biphenyl/polychlorinated biphenyls (PCBs) in the microbe. To elucidate detailed structures of BphC reaction intermediates, crystal structures of the substrate-free form, the BphC-substrate complex, and the BphC-substrate-NO (nitric oxide) complex were determined. These crystal structures revealed (1) the binding site of the O(2) molecule in the coordination sphere and (2) conformational changes of His194 during the catalytic reaction. On the basis of these findings, we propose a catalytic mechanism for the extradiol-cleaving catecholic dioxygenase in which His194 seems to play three distinct roles. At the early stage of the catalytic reaction, His194 appears to act as a catalytic base, which likely deprotonates the hydroxyl group of the substrate. At the next stage, the protonated His194 seems to stabilize a negative charge on the O2 molecule located in the hydrophobic O2-binding cavity. Finally, protonated His194 seems to function as a proton donor, whose existence has been proposed.     

Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy

Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.     

TorsinA protein degradation and autophagy in DYT1 dystonia

Early-onset generalized dystonia (DYT1) is a debilitating neurological disorder characterized by involuntary movements and sustained muscle spasms. DYT1 dystonia has been associated with two mutations in torsinA that result in the deletion of a single glutamate residue (torsinA �”E) and six amino-acid residues (torsinA �”323-8). We recently revealed that torsinA, a peripheral membrane protein, which resides predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope (NE), is a long-lived protein whose turnover is mediated by basal autophagy. Dystonia-associated torsinA �”E and torsinA �”323-8 mutant proteins show enhanced retention in the NE and accelerated degradation by both the proteasome and autophagy. Our results raise the possibility that the monomeric form of torsinA mutant proteins is cleared by proteasome-mediated ER-associated degradation (ERAD), whereas the oligomeric and aggregated forms of torsinA mutant proteins are cleared by ER stress-induced autophagy. Our findings provide new insights into the pathogenic mechanism of torsinA �”E and torsinA �”323-8 mutations in dystonia and emphasize the need for a mechanistic understanding of the role of autophagy in protein quality control in the ER and NE compartments.

Addendum to: Giles LM, Chen J, Li L, Chin L-S. Dystonia-associated torsinA mutations cause premature degradation of torsinA protein and cell-type-specific mislocalization to the nuclear envelope. Hum Mol Genet 2008; 17:2712-22; PMID: 18552369; DOI: 10.1093/hmg/ddn173.     

Redox potentials of the oriented film of the wild-type,the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were -470 mV for the 13-cis configuration of the retinal Shiff base in bR and -757 mV for the all-trans configuration in H(2)O, and -433 mV for the 13-cis configuration and -742 mV for the all-trans configuration in D(2)O. The solvent isotope effect (DeltaV=V(D(2)O)-V(H(2)O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated C=N part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were -507 mV for the 13-cis configuration and -788 mV for the all-trans configuration; and for the E204Q mutant they were -491 mV for the 13-cis configuration and -769 mV for the all-trans configuration. Replacement of the Glu(194) or Glu(204) residues by Gln weakened the electron withdrawing interaction to the protonated C=N bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were -471 mV for the 13-cis configuration and -760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the C=N part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.     

LncRNA H19/miR-194/PFTK1 axis modulates the cell proliferation and migration of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains a huge challenge due to its high mortality and morbidity; gene therapy might be a promising treatment for PDAC. The critical role of Wnt-signaling pathway in cancer pathogenesis has been widely recognized; cyclin-dependent kinase 14 (CDK14, PFTK1)-induced low-density lipoprotein receptor-related proteins 5/6 (LRP5/6) phosphorylation is an important issue in Wnt-signaling activation. Long noncoding RNA (LncRNA)-microRNA (miRNA)-messenger RNA (mRNA) modulating the pathogenesis of cancers has been regarded as a major mechanism. In the current study, upregulated lncRNAs positively correlated with PFTK1 were analyzed and selected using The Cancer Genome Atlas (TCGA) database. Of them, lncRNA H19 can activate Wnt signaling in cancers. In PDAC tissues, the expression of H19 and PFTK1 were upregulated; H19 knockdown suppressed the cell proliferation and migration of PDAC, while PFTK1 overexpression partially attenuated the suppressive effect of H19 knockdown. As analyzed by TCGA and predicted by online tools, miR-194 was negatively correlated with PFTK1 and might bind to both H19 and PFTK1, which was further confirmed by luciferase reporter and RNA immunoprecipitation assays. Moreover, the effect of H19 knockdown on PFTK1 protein and the cell proliferation and migration could be partially reversed by miR-194 inhibition; H19/miR-194 axis modulated PDAC cell proliferation and migration through PFTK1 downstream Wnt signaling. Results suggested that rescuing miR-194 expression in PDAC can inhibit lncRNA H19 and PFTK1 expression, subsequently suppressing PDAC cell proliferation and migration. Due to the complexity of the lncRNA-miRNA-mRNA network, further in vivo experiments examining potential side effects are needed in future study to explore the clinical application of these findings.     

Cloning and analysis of the MAT1-2-1 gene from the traditional Chinese medicinal fungus Ophiocordyceps sinensis

The entomopathogenic fungus Ophiocordyceps sinensis has been important in traditional Chinese medicine but has yet to be commercially cultivated. One bottleneck is the very low frequency of stromata formation from artificially infected moth larvae. The mating system of fungi is the determining factor for sexual reproduction, but mating-type genes of O. sinensis have not been previously investigated. In this study, the putative mating-type gene MAT1-2-1 within the MAT1-2 idiomorph was amplified by polymerase chain reaction (PCR) and was determined to consist of 859 nucleotides that encode 249 amino acids; genes within the MAT1-1 idiomorph, however, were not determined. The MAT1-2-1 gene contained the conserved high-mobility group (HMG) box, and MAT1-2-1 flanking sequences were subsequently obtained. Although no putative open reading frames of the MAT1-1 idiomorph were detected within the ca. 8-kb flanking sequences of MAT1-2-1, a putative DNA lyase gene (which is present next to both idiomorphs in some heterothallic fungi) was found ca. 3.0 kb downstream of MAT1-2-1. The intervening distance between MAT1-2-1 and the DNA lyase gene in O. sinensis is larger than that in Cordyceps militaris and Cordyceps takaomontana. In addition, O. sinensis showed low sequence similarities with C. militaris and C. takaomontana in both MAT1-2-1 and the DNA lyase gene. In the phylogenetic tree, different MAT1-2-1 haplotypes of O. sinensis clustered together with high bootstrap support. As a single-copy gene, MAT1-2-1 was detected in all examined O. sinensis isolates including tissue cultures and single-ascospore cultures. This report describes, for the first time, a mating-type gene of O. sinensis.     

Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).     

Analysis of the polymorphisms XRCC1Arg194Trp and XRCC1Arg399Gln in gliomas

XRCC genes (X-ray cross-complementing group) were discovered mainly for their roles in protecting mammalian cells against damage caused by ionizing radiation. Studies determined that these genes are important in the genetic stability of DNA. Although the loss of some of these genes does not necessarily confer high levels of sensitivity to radiation, they have been found to represent important components of various pathways of DNA repair. To ensure the integrity of the genome, a complex system of DNA repair was developed. Base excision repair is the first defense mechanism of cells against DNA damage and a major event in preventing mutagenesis. Repair genes may play an important role in maintaining genomic stability through different pathways that are mediated by base excision. In the present study, we examined XRCC1Arg194Trp and XRCC1Arg399Gln polymorphism using PCR-RFLP in 80 astrocytoma and glioblastoma samples. Patients who had the allele Trp of the XRCC1Arg194Trp polymorphism had an increased risk of tumor development (OR = 8.80; confidence interval at 95% (95%CI) = 4.37-17.70; P < 0.001), as did the allele Gln of XRCC1Arg399Gln (OR = 1.01; 95%CI = 0.53-1.93; P = 0.971). Comparison of overall survival of patients did not show significant differences. We suggest that XRCC1Arg194Trp and XRCC1Arg399Gln polymorphisms are involved in susceptibility for developing astrocytomas and glioblastomas.     

Rad24 truncation,coupled with altered telomere structure,promotes cdc13-1 suppression in S. cerevisiae

Distinguishing telomeres from DNA double strand breaks is critical for genome stability. In S. cerevisiae, the Cdc13 single-strand telomere binding protein is critical for protecting chromosome ends. The C-rich telomere strand is lost at high temperatures in cdc13-1 strains, leading to activation of the DNA damage checkpoint and cell inviability. Through a screen performed to identify activities involved in telomere C-strand loss, we identified two new rad24 alleles. Rad24 is an alternate Rfc1 subunit, functioning to load the 9-1-1 checkpoint clamp. In each rad24 allele, a transposon inserted within the RAD24 coding region leads to expression of different carboxyl-terminal portions of Rad24, deleting or truncating the amino-terminus. We show that an intact Rad24 amino-terminus is necessary for its checkpoint function. Interestingly, the initial cdc13-1 rad24-2 strains grew at 36Ã?Â?Å¡C, but the extent of suppression associated with rad24-2 weakened in serial backcrosses, and cdc13-1 segregants from these crosses showed a modest increase in temperature resistance. Moreover, while a RAD24 plasmid suppressed the checkpoint defect in the initial cdc13-1 rad24-2 strain, the temperature resistance was only partially suppressed. These data suggest that the TG1-3 amplification observed in this strain contributes to the suppression phenotype. By recreating the rad24-2 allele in a strain with normal telomeres, we find that, relative to the rad24-Ã?¢Ã?Â?†allele, rad24-2 increases the frequency of obtaining cdc13-1 cells capable of growth at high temperatures. Our hypothesis is that the Rad24-2 truncation protein affects telomere structure or recombination in a manner distinct from rad24-Ã?¢Ã?Â?†.     

A gene controlling segregation of the Bacillus subtilis plasmid pC194

Summary Plasmid pC194-1, a mutant of pC194, and chimeric derivatives of pC194-1 are segregationally unstable in B. subtilis. Such instability could be enhanced by exposure of pC194-1-carrying cells to methyl methanesulfonate. pC194-1 is distinct from pC194 in the addition of two A:T base pairs within the previously defined D region of pC194. Complementation experiments between pC194-1 and other plasmids suggest that the mutation of pC194-1 interferes with the production of a diffusible gene product required for plasmid maintenance.     

Investigating interdomain region mutants Phe194----Leu and Phe194----Trp of yeast phosphoglycerate kinase by 1H-NMR spectroscopy.

Site-directed mutagenesis has been used to produce two mutant forms of yeast phosphoglycerate kinase in which the interdomain residue Phe194 has been replaced by a leucine or tryptophan residue. Using 1H-NMR spectroscopy, it was found that the mutations at position 194 induce both local and long-range conformational changes in the protein. It was also found that 3-phosphoglycerate binding to the mutant proteins induces somewhat different conformational effects to those observed for wild-type phosphoglycerate kinase. The affinity of mutant Phe194----Trp for 3-phosphoglycerate was found by NMR studies to be unaffected, while the affinity of Phe194----Leu mutant is reduced by about threefold relative to the wild-type enzyme. The binding of ATP at the electrostatic site of the mutant proteins is also seen to be about three times weaker for the Phe194----Leu mutant when compared to wild-type or Phe194----Leu mutant. These results are discussed in the light of the kinetic studies on the mutants which show that for Phe194----Leu mutant the Km values for both 3-phosphoglycerate and ATP, as well as the Vmax, are decreased relative to the wild-type enzyme, while for mutant Phe194----Trp, the Km values for 3-phosphoglycerate and ATP are unaffected and the Vmax is decreased when compared to wild-type enzyme. Kinetic studies in the presence of sulphate reveal that the anion activation is greater for mutant Phe194----Trp and less for mutant Phe194----Leu, relative to that observed for wild-type phosphoglycerate kinase. The NMR data, taken together with the kinetic data, are consistent with the on and off rates of 3-phosphoglycerate being affected by the mutations at position 194. It is suggested that Phe194 is important for the mobility of the interdomain region and the relative movement of the 3-phosphoglycerate binding site which allows the optimum conformation for catalysis to be attained. Apparently Trp194 reduces the mobility of the interdomain region of the protein, while Leu194 increases it.     

Establishing irremediable psychiatric suffering in the context of medical assistance in dying in the Netherlands: a qualitative study

Background:Establishing irremediability of suffering is a central challenge in determining the appropriateness of medical assistance in dying (MAiD) for patients with a psychiatric disorder. We sought to evaluate how experienced psychiatrists define irremediable psychiatric suffering in the context of MAiD and what challenges they face while establishing irremediable psychiatric suffering.Methods:We conducted a qualitative study of psychiatrists in the Netherlands with experience assessing irremediable psychiatric suffering in the context of MAiD. We collected data from in-depth, semistructured interviews focused on the definition of irremediable psychiatric suffering and on the challenges in establishing irremediability. We analyzed themes using a modified grounded theory approach.Results:The study included 11 psychiatrists. Although irremediable psychiatric suffering is a prospective concept, most participants relied on retrospective dimensions to define it, such as a history of failed treatments, and expressed that uncertainty was inevitable in this process. When establishing irremediable psychiatric suffering, participants identified challenges related to diagnosis and treatment. The main diagnostic challenge identified was the frequent co-occurrence of more than 1 psychiatric diagnosis. Important challenges related to treatment included assessing the quality of past treatments, establishing when limits of treatment had been reached and managing “treatment fatigue.”Interpretation:Challenges regarding the definition, diagnosis and treatment of irremediable psychiatric suffering complicate the process of establishing it in the context of MAiD. Development of consensus clinical criteria for irremediable psychiatric suffering in this context and further research to understand “treatment fatigue” among patients with psychiatric disorders may help address these challenges. Registration: This study was preregistered under osf.io/2jrnd.

Medical assistance in dying (MAiD), also known as physician-assisted death, has been legalized in an increasing number of jurisdictions around the world.¹ In 2023, Canada will join a small group of countries that allow MAiD for people with mental illness, more commonly referred to as persons with a psychiatric disorder. ² In the Netherlands, MAiD for irremediable psychiatric suffering has been approved by jurisprudence since the 1990s and it has been regulated by law since 2002. The last decade has seen a marked increase in MAiD for irremediable psychiatric suffering; in 2020, MAiD was performed 88 times for psychiatric suffering (1.3% of all MAiD cases), compared with just 2 instances in 2010.³ The number of requests by patients with a psychiatric disorder is much higher, but 90% of requests do not end in MAiD. Sometimes, they are retracted by patients, but most are denied by psychiatrists.⁴The main legal requirements for MAiD in the Netherlands are that the patient must be able to make a competent request, that the patient’s suffering must be unbearable and irremediable, and that the patient and physician agree that there are no other reasonable treatment options. The process for MAiD requires an assessment by an independent physician and, in the case of psychiatric suffering, a third assessment by an independent psychiatrist, preferably one with specific expertise regarding the patient’s disorder.⁵ The Canadian legal requirements under the amended Bill C-7, which will come into effect in 2023, will be largely similar to the Dutch requirements.⁶ However, Canadian legislation, which does not yet permit MAiD for mental illness, has 1 important difference: patients with decision-making capacity can qualify for MAiD if they refuse treatments that they do not find acceptable. It is as yet unknown whether this approach to refusal of treatment will also be applied to MAiD where mental illness is the sole underlying condition.Although there are concerns about decision-making capacity, the central dilemma of MAiD for patients with a psychiatric disorder appears to revolve around applying the concept of irremediability to psychiatric disorders. The 2018 guideline by the Dutch Psychiatry Association defines irremediable psychiatric suffering in the context of MAiD as follows: “irremediability means that there is no longer any prospect of alleviating, mitigating, enduring or removing suffering. There is no longer a reasonable treatment perspective.”⁷ Elsewhere, the guideline states that reasonable treatment perspective means that “there is a prospect of improvement with adequate treatment, within a foreseeable period, and with a reasonable ratio between the expected results and the burden of the treatment for the patient.”⁷A recent scoping review identified a multitude of conceptual articles addressing irremediability in the context of psychiatric MAiD but few empirical studies.¹ This suggests that the assessment of irremediable psychiatric suffering is particularly difficult relative to suffering arising from other types of conditions.Surveys estimate that 46% of psychiatrists in the Netherlands have received an explicit MAiD request at least once in their career, and 4% actually assisted in the death of a patient with a psychiatric disorder.⁸ The experiences of psychiatrists who have handled MAiD requests can be seen as an important source of knowledge about the challenges of establishing irremediable psychiatric suffering in practice. The aim of this study was to learn how experienced psychiatrists define psychiatric suffering as irremediable in the context of a MAiD request and what challenges they face while establishing irremediable psychiatric suffering.     

Silencing circ-USP1 protects the renal tubular from kidney injury induced by hypoxia via modulating miR-194-5p/DNMT3A axis in acute renal allografts

Recent studies indicate that circular RNAs are involved in dysregulation of kidney injury. Nevertheless, the underlying mechanisms remain largely unclear. Therefore, this study sought to investigate the role of circ-USP1 in the pathogenesis of early renal allografts. Thirty-two male C57BL/6J mice aged between 6 and 8 weeks were divided into the sham and allograft groups. Thereafter, the association between miR-194-5p, circ-USP1 and DNMT3A was confirmed using a combination of bioinformatics and the luciferase reporter gene assay. Additionally, the expression of circ-USP1, miR-194-5p and DNMT3A mRNA was detected through qPCR. Afterwards, the Western blot assay was performed to examine the expression of DNMT3A protein. Finally, the TUNEL assay was conducted to determine the rate of apoptosis in DNMT3A cells. The expression of circ-USP1 increased, while that of miR-194-5p decreased in renal allografts. Additionally, silencing circ-USP1 reduced kidney injuries caused by renal allografts in mice. Moreover, miR-194-5p was a target for circ-USP1, and DNMT3A was a target of miR-194-5p. Finally, it was shown that silencing circ-USP1 reduced DNMT3A expression in the kidney of mice that received renal allografts. Circ-USP1 functions as a competing endogenous RNA for miR-194-5p. This occurs in order to regulate DNMT3A expression in kidney injury induced by hypoxia in acute renal allografts.     

Plasmid transduction by Bacillus subtilis bacteriophage SPP1: effects of DNA homology between plasmid and bacteriophage.

Any SPP1 DNA restriction fragment cloned into Bacillus subtilis plasmid pC194 or pUB110 increased the transduction frequency of the plasmid by SPP1 100- to 1,000-fold over the transduction level of the plasmid alone. This increment was observed irrespective of whether a fragment contained the SPP1 packaging origin (pac). Furthermore, an SPP1 derivative into whose genome pC194 DNA had been integrated transduced pC194 DNA with a greatly enhanced frequency. Transduction enhancement mediated by DNA-DNA homology between plasmid and SPP1 was independent of the extent of homology (size range analyzed, 0.5 to 3.9 kilobases) and the recombination proficiency of donor or recipient.     

Polymorphisms of DNA base‐excision repair genes APE/Ref‐1 and XRCC1 are not associated with the risk for Graves' disease

Oxidative stress has been implicated in etiopathogenesis of Graves' disease (GD). Increased lipid peroxidation and oxidative DNA damage have been found in GD patients. Oxidative DNA damage is mainly repaired by the base‐excision repair (BER) pathway. Polymorphisms in DNA‐repair genes have been associated with the increased risk of various diseases and could also be related to the etiology of GD. Therefore, we conducted a study including 197 patients with GD and age‐ and sex‐matched 303 healthy subjects to examine the role of single‐nucleotide polymorphisms of BER genes, APE/Ref‐1 (codon 148) and XRCC1 (codons 194 and 399) as a risk factor for GD. These polymorphisms were determined by quantitative real‐time PCR and melting curve analysis using LightCycler. No significant association was observed between the variant alleles of APE/Ref‐1 codon 148 [odds ratio (OR) = 0.89, 95% confidence interval (CI) = 0.69–1.17], XRCC1 codon 194 (OR = 1.24, 95% CI = 0.79–1.94), and XRCC1 codon 399 (OR = 1.12, 95% CI = 0.86–1.46) and GD. These preliminary results suggest that APE/Ref‐1 (codon 148) and XRCC1 (codons 194 and 399) polymorphisms are not significant risk factors for developing GD. Copyright © 2009 John Wiley & Sons, Ltd.