Development and clinical validation of a dual ddPCR assay for detecting carbapenem-resistant Acinetobacter baumannii in bloodstream infections.

Objective: Acinetobacter baumannii (A. baumannii, AB) represents a major species of Gram-negative bacteria involved in bloodstream infections (BSIs) and shows a high capability of developing antibiotic resistance. Especially, carbapenem-resistant Acinetobacter baumannii (CRAB) becomes more and more prevalent in BSIs. Hence, a rapid and sensitive CRAB detection method is of urgent need to reduce the morbidity and mortality due to CRAB-associated BSIs. Methods: A dual droplet digital PCR (ddPCR) reaction system was designed for detecting the antibiotic resistance gene OXA-23 and AB-specific gene gltA. Then, the specificity of the primers and probes, limit of detection (LOD), linear range, and accuracy of the assay were evaluated. Furthermore, the established assay approach was validated on 37 clinical isolates and compared with blood culture and drug sensitivity tests. Results: The dual ddPCR method established in this study demonstrated strong primer and probe specificity, distinguishing CRAB among 21 common clinical pathogens. The method showed excellent precision (3 × 10-4 ng/µL, CV < 25%) and linearity (OXA-23: y = 1.4558x + 4.0981, R2 = 0.9976; gltA: y = 1.2716x + 3.6092, R2 = 0.9949). While the dual qPCR LOD is 3 × 10-3 ng/µL, the dual ddPCR's LOD stands at 3 × 10-4 ng/µL, indicating a higher sensitivity in the latter. When applied to detect 35 patients with BSIs of AB, the results were consistent with clinical blood culture identification and drug sensitivity tests. Conclusion: The dual ddPCR detection method for OXA-23 and gltA developed in this study exhibits good specificity, excellent linearity, and a higher LOD than qPCR. It demonstrates reproducibility even for minute samples, making it suitable for rapid diagnosis and precision treatment of CRAB in BSIs.

The variation of antigenic and histo-blood group binding sites synergistically drive the evolution among chronologically emerging GII.4 noroviruses.

Norovirus, commonly found on shellfish and vegetables, is a foodborne virus with GII.4 as the dominant genotype responsible for widespread outbreaks since 1995. Continuous variation of major capsid protein VP1 can lead to changes in the immunogenicity and host receptor binding ability of norovirus, which is an important evolutionary mechanism. Therefore, analyzing the immunogenicity of VP1 and its binding ability to various HBGAs in GII.4 variants could improve our understanding of the persistent prevalence of GII.4. Here, the results suggest that GII.4 has gradually enhanced its HBGAs binding ability over time for various types of receptors. Variants exhibit significantly stronger immune response to homologous mouse antiserum than heterologous ones, highlighting the importance of variation of antigenic and histo-blood group binding sites in driving the evolution of GII.4. These synergistic forces constantly lead to antigenic drift and changes in receptor binding, resulting in continuous emergence of new variant strains and sustained prevalence.

Complete remission of gallbladder neuroendocrine carcinoma with liver metastasis by tislelizumab plus chemotherapy: a case report.

Background: Gallbladder neuroendocrine carcinoma (GB-NEC) is an extremely rare cancer with a poor prognosis in the clinic. Although surgical resection remains the primary and preferred therapeutics, many patients are in a late stage and lose the opportunity for surgery. However, due to the extremely low morbidity, the specific treatment guidelines for GB-NEC have not been established. Case presentation: A 52-year-old woman was admitted to our hospital with the chief complaint of "almost 1 month after palliative surgery for metastatic gallbladder carcinoma." According to the results of pathological findings and imaging manifestations, the patient was diagnosed with GB-NEC with a clinical stage of pT3N1M1 (IVB). The patient then received tislelizumab plus EP chemotherapy (etoposide 100 mg + cisplatin 30 mg, d1-3) every 3 weeks for 8 cycles from 12 November, 2021, followed by maintenance therapy (tislelizumab alone) every 3 weeks until now. The tumor response was evaluated as complete remission since 13 February, 2023. As of the last follow-up, the patient remains alive, with no complaints of discomfort. Conclusions: Gallbladder NEC has no specific symptoms, and the diagnosis is based on pathological and immunohistochemical results. The therapeutic course and efficacy of the case in this study indicates that the application of PD-1 inhibitor might be a feasible therapeutic option for GB-NEC. However, this potential strategy needs validation by further clinical studies in the future.

Sensory circuitry controls cytosolic calcium-mediated phytochrome B phototransduction.

Although Ca2+ has long been recognized as an obligatory intermediate in visual transduction, its role in plant phototransduction remains elusive. Here, we report a Ca2+ signaling that controls photoreceptor phyB nuclear translocation in etiolated seedlings during dark-to-light transition. Red light stimulates acute cytosolic Ca2+ increases via phyB, which are sensed by Ca2+-binding protein kinases, CPK6 and CPK12 (CPK6/12). Upon Ca2+ activation, CPK6/12 in turn directly interact with and phosphorylate photo-activated phyB at Ser80/Ser106 to initiate phyB nuclear import. Non-phosphorylatable mutation, phyBS80A/S106A, abolishes nuclear translocation and fails to complement phyB mutant, which is fully restored by combining phyBS80A/S106A with a nuclear localization signal. We further show that CPK6/12 function specifically in the early phyB-mediated cotyledon expansion, while Ser80/Ser106 phosphorylation generally governs phyB nuclear translocation. Our results uncover a biochemical regulatory loop centered in phyB phototransduction and provide a paradigm for linking ubiquitous Ca2+ increases to specific responses in sensory stimulus processing.

Identification of a Two-lncRNA Signature with Prognostic and Diagnostic Value for Hepatocellular Carcinoma.

Background: Accumulating evidence has revealed the important role of long noncoding RNAs (lncRNA) in tumorigenesis and progression of hepatocellular carcinoma (HCC). This study aimed to identify potential lncRNAs that can serve as diagnostic and prognostic signatures for HCC. Methods: Expression profiling analysis was performed to identify differentially expressed lncRNAs (DElncRNA) between HCC and matched normal samples by integrating two independent microarray datasets. Functional Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were explored by Gene Set Variation Analysis. The prognostic and diagnostic models were developed based on two DElncRNAs. Real-time PCR was used to quantify the relative expressions of candidate lncRNAs. Results: Two robust DElncRNAs were identified and verified by quantitative PCR between HCC and matched normal samples. Function enrichment analysis revealed that they were associated with the wound healing process. The two lncRNAs were subsequently used to construct a prognostic risk model for HCC. Patients with high-risk scores estimated by the model showed a shorter survival time than low-risk patients (P < 0.001). Besides, the two lncRNA-based HCC diagnostic models exhibited good performance in discriminating HCC from normal samples on both training and test sets. The values of area under the curve (AUC) for early (I-II) and late (III-IV) HCC detection were 0.88 and 0.93, respectively. Conclusions: The two wound healing-related DElncRNAs showed robust performance for HCC prognostic prediction and detection, implying their potential role as diagnostic and prognostic markers for HCC.

Genomic mutation characteristics and prognosis of biliary tract cancer.

BACKGROUND: The incidence of biliary system cancer is higher in the Chinese population than in the West. The overall prognosis of gallbladder cancer and cholangiocarcinoma is poor, and the current treatment is limited. In order to explore the pathogenesis of biliary tract cancers and potential targeted therapies, we mapped the mutation landscape of biliary tract cancer in the Chinese population and analyzed the molecular mechanism related to prognosis. METHODS: A total of 59 formalin fixed paraffin-embedded (FFPE) tissue samples were obtained from patients with operable biliary tract cancer. We conducted targeted capture sequencing of 620 genes through high-throughput sequencing technology and analyzed the fusion information of 13 genes. RESULTS: Mutations were detected in 88% samples, and the most frequent mutation base was C>T. Genes with higher single nucleotide variations (SNV) and copy number variations (CNV) frequency are TP53, KRAS, ARID1A, VEGFA, cyclin family related genes and cyclin-dependent kinase genes. Actionable mutations were detected in 59.3% samples, and germline mutations were detected in 22% samples. Patients with KRAS mutations, VEGFA pathway mutations and higher tumor mutation burden (TMB) may have poor prognosis. CONCLUSIONS: We explored the mutation characteristics and prognostic mechanism of biliary tract cancers in the Chinese population. This study provides potential evidence for targeted therapy and immunotherapy of biliary tract cancers.

Rapid screening for antigenic characterization of GII.17 norovirus strains with variations in capsid gene.

The emergence of the novel GII.17 Kawasaki 2014 norovirus variant raising the interest of the public, has replaced GII.4 as the predominant cause of noroviruses outbreaks in East Asia during 2014-2015. Antigenic variation of the capsid protein is considered as one of the key mechanisms of norovirus evolution. In this study, we screened a panel of GII.17 mutants. First, we produced norovirus P proteins using cell-free protein synthesis (CFPS) system, comparing the results to pure proteins expressed in a cell-based system. Next, we determined the binding capability of specific monoclonal antibody (mAb) 2D11 using a unique set of wild-type GII.17 strains. Results of the EIA involving a panel of mutant cell-free proteins indicated that Q298 was the key residue within loop 1. These data highlighted the essential residues in the linear antibody binding characteristics of novel GII.17. Furthermore, it supported the CFPS as a promising tool for rapidly screening mutants via the scalable expression of norovirus P proteins.

Epochal coevolution of minor capsid protein in norovirus GII.4 variants with major capsid protein based on their interactions over the last five decades.

Norovirus is a leading cause of viral gastroenteritis outbreaks worldwide, with GII.4 responsible for the majority of infections. Minor capsid protein VP2 has been found to have functions such as stabilizing virus particles, and VP2 is one of the highly variable proteins of norovirus, similar to major capsid protein VP1. However, whether the variation of VP2 is functionally driven still remains unclear. In this study, VP2 showed a higher evolutionary rate (2.642×10-3 substitutions/site/year) than VP1 (1.587×10-3 substitutions/site/year), and a hypervariable region in VP2 in a serial of norovirus GII.4 over the past 50 years had been observed. Notably, the high variation of VP2 was not haphazard. The evolutionary process of VP2 is similar to that of VP1 with comparable topologies when the phylogenetic trees were constructed. Moreover, VP2 was found to interact with VP1 among epidemic variants of GII.4 using the yeast two-hybrid experiments. The results of interactions were grouped into time-adjacent (e.g. Ancestral-VP1 plus US95-VP2) and non-adjacent (e.g. Ancestral-VP1 plus Sydney-VP2) according to the epochal chronologically based prevalence of GII.4 norovirus. Interestingly, the interaction of the former group was significantly stronger than that of the latter group (P=0.0001). Furthermore, the interaction regions on VP2 (residues 131-160 and 171-180) were mapped to the hypervariable region. And these interaction regions did show an important role in the evolutionary process of VP2, which was consistent with that of VP1. In summary, the minor capsid protein VP2 of GII.4 noroviruses had shown the epochal coevolution with VP1 based on their interactions over the past 50 years. The findings of this study provided valuable information for further understanding and completing the evolutionary mechanism of norovirus.

Integration of light and temperature sensing by liquid-liquid phase separation of phytochrome B.

Light and temperature in plants are perceived by a common receptor, phytochrome B (phyB). How phyB distinguishes these signals remains elusive. Here, we report that phyB spontaneously undergoes phase separation to assemble liquid-like droplets. This capacity is driven by its C terminus through self-association, whereas the intrinsically disordered N-terminal extension (NTE) functions as a biophysical modulator of phase separation. Light exposure triggers a conformational change to subsequently alter phyB condensate assembly, while temperature sensation is directly mediated by the NTE to modulate the phase behavior of phyB droplets. Multiple signaling components are selectively incorporated into phyB droplets to form concentrated microreactors, allowing switch-like control of phyB signaling activity through phase transitions. Therefore, light and temperature cues are separately read out by phyB via allosteric changes and spontaneous phase separation, respectively. We provide a conceptual framework showing how the distinct but highly correlated physical signals are interpreted and sorted by one receptor.

The VP2 protein exhibits cross-interaction to the VP1 protein in norovirus GII.17.

Norovirus is a major cause of acute gastroenteritis worldwide. Like the major capsid protein (VP1), the minor capsid protein (VP2) also contains a hypervariable domain. Generally, a hypervariable domain is functionally driven. However, many functions of VP2 remain unknown and worth exploring. Without sufficient sequences and an available crystallographic model, it is difficult to explore VP2's mysteries. As a helper of stabilizing and coordinating the formation of virus-like particles (VLPs), we asked whether VP2 interacted with the major capsid protein (VP1) in GII.17 and if so, what the key interaction residues were. Here, we reported cross-interaction among four strains represented four clusters of GII.17, and the VP1 interaction domain of VP2 (174-179aa) was found. However, the VP1 interaction domain of VP2 was not universal in different clusters of GII.17. VP2 might evolve in a different pattern from VP1. Additionally, in contrast to previous reports, we found that VP2 localized in the cytoplasm. More possibilities of VP2 should be further explored.

Association of fucosyltransferase 2 gene with norovirus infection: A systematic review and meta-analysis.

BACKGROUND: Norovirus is a leading cause of viral gastroenteritis outbreaks worldwide. Histo-blood group antigens (HBGAs) are important host attachment factors in susceptibility to norovirus. In this study, the association of FUT2 gene, which participates in the biosynthesis of HBGAs, with norovirus infection has been investigated. METHODS: All relevant studies on the associations of FUT2 gene with norovirus were retrieved from PubMed, Web of Science, Embase, and Cochrane Library databases. Odds ratios (ORs) and 95% confidence interval (CI) were used to analyze the extracted data. I2 statistic, sensitivity analysis and publication bias analysis were used to confirm the findings. Subgroup analyses were performed for races, genotypes, development degree of the countries, publication years, age and setting when heterogeneity was recorded. RESULTS: Twenty studies including 4066 participants were included for the meta-analysis. This analysis showed that there is a significant association between FUT2 gene and norovirus infection (OR = 3.02, 95%CI = 2.00-4.55, P < 0.001). Additionally, the ORs of norovirus infection among Chinese (OR = 4.49, 95%CI = 2.37-8.50, P < 0.001) were higher than those among Caucasian (OR = 3.23, 95%CI = 2.20-4.74, P < 0.001). CONCLUSIONS: The meta-analysis suggested that FUT2 gene is associated with susceptibility to norovirus infection.

Global prevalence of norovirus in cases of acute gastroenteritis from 1997 to 2021: An updated systematic review and meta-analysis.

BACKGROUND: The worldwide response towards the acute gastroenteritis epidemic was well known, but the absence of an updated systematic review of global norovirus epidemiology in cases of gastroenteritis existed. We aimed to conduct and update a systematic review and meta-analysis of studies assessing norovirus prevalence among gastroenteritis patients worldwide. METHODS: Four databases (PubMed, EMBASE, Cochrane Library, and Web of Science) were searched for epidemiological papers from 2014 to 2021 which applied the PCR method to access the prevalence of norovirus in acute gastroenteritis patients more than a full year. Statistical analysis was conducted using R-4.0.0 software. RESULTS: A total of 405 records with 842, 926 cases were included. The pooled prevalence of norovirus was 16% (95%CI 15, 17). Children under 5 years old were at a higher risk with norovirus. A higher prevalence was seen in South America (22%, 95% CI 18, 27), while other continents showed a similar result with the overall prevalence of norovirus. No association was found between national income level and norovirus prevalence. A gradient of decreasing prevalence was noticed from community (20%, 95% CI 16, 24) to outpatients (18%, 95% CI 16, 20) to hospital setting (included both in- and outpatients, 17%, 95% CI 16, 19) to inpatients (15%, 95% CI 13, 17). CONCLUSION: Norovirus were associated with 16% acute gastroenteritis globally. To fully understand the prevalence of norovirus worldwide, the continual surveillance of norovirus epidemics was required.

ABO blood group-associated susceptibility to norovirus infection: A systematic review and meta-analysis.

BACKGROUND: Norovirus is responsible for the viral gastroenteritis burden worldwide. Histo-blood type antigens (HBGAs) are the only well-known factor regarding their effect on the pathogenesis of norovirus. Here, we performed the study to further investigate the association of the ABO blood group with norovirus susceptibility. METHODS: All relevant studies were retrieved from PubMed, Embase, Web of Science, and Cochrane Library databases and the associations of ABO blood groups with norovirus were assessed. The pooled odds ratios (ORs) and 95% confidence interval (CI) were calculated from extracted data. I2 statistics, sensitivity analysis, and publication bias were used to confirm the findings. Subgroup analyses were performed for genotypes, publication years, development degree of the countries, and age if heterogeneity was recorded. RESULTS: Seventeen articles covering 2304 participants were included. The overall analysis of the studies showed similar ORs of norovirus infection among individuals with blood type A (OR = 0.90, 95%CI = 0.71-1.14, P = 0.37) and blood type B (OR = 0.85, 95%CI = 0.66-1.12, P = 0.25) as compared to those controls. An increased odds of norovirus infection was found among individuals with blood type O (OR = 1.28, 95%CI = 1.03-1.59, P = 0.03), while the individuals with blood type AB (OR = 0.91, 95%CI = 0.60-1.39, P = 0.67) showed no correlation with norovirus infection. For blood type B and blood type AB, the results of subgroup analyses mirrored the observations above. CONCLUSIONS: The meta-analysis suggested that the blood type A, B and AB might not affect susceptibility to norovirus infection. However, blood type O appeared to be more susceptible to norovirus infection.

Diagnostic test accuracy of droplet digital PCR for the detection of EGFR mutation (T790M) in plasma: Systematic review and meta-analysis.

BACKGROUND: T790M mutation was a primary lead cause in the acquired resistance to EGFR-TKIs confirmed in earlier studies. Since the shortcomings of tumor tissue detection are well known, the liquid biopsy is more appropriate to track T790M status. We assessed the accuracy and clinical significance of the droplet digital PCR (ddPCR) detection of T790M mutation in plasma. METHODS: We retrieved PubMed, Embase, Cochrane, and Web of science with no limitation of language and publication year. Summary sensitivity and specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio of detection of EGFR T790M status were calculated from extracted data from included articles. The summary receiver operating curve (SROC), diagnostic odds ratio (DOR), and the area under the summary receiver operating curve (AUC) was used to assess the overall diagnostic accuracy. I2 and meta-regression were used to evaluate heterogeneity and the source of heterogeneity, respectively. RESULT: We identified 15 studies in the total search of 1364 reports, including 427 paired tissue and plasma samples. The pooled sensitivity and the pooled specificity were 0.68 (95% CI 0.61-0.75) and 0.85 (95% CI 0.75-0.91) by the bivariate model, respectively. The AUC and the pooled DOR were 0.78 (95% CI 0.74-0.81) and 12 (95% CI 7-22), respectively. None of the cofactors could account for the heterogeneity. CONCLUSION: The plasma analysis is of a promising performance to screen EGFR-T790M mutation status by ddPCR.

Oligomerization and Photo-Deoligomerization of HOOKLESS1 Controls Plant Differential Cell Growth.

Apical hook curvature is crucial for buried seedling survival and a superb model for dissecting differential cell growth. HOOKLESS1 (HLS1) is essential for apical hook formation, acting as a hub integrating various external and internal signals. However, its functional mechanism remains unclear. Here, we demonstrate that HLS1 protein is present as an oligomer in the nucleus of dark-grown seedlings. Oligomerization is required for HLS1 activation, as the mutated HLS1 protein abolishing self-association exists as nonfunctional monomers. Upon light exposure, photoreceptor phyB translocates into the nucleus and interacts with HLS1, disrupting the self-association and oligomerization of HLS1 to initiate hook unfolding. Remarkably, genetic expression of nuclear-localized phyB is sufficient to inactivate HLS1, resulting in compromised hook curvature in etiolated seedlings. Together, we conclude that HLS1 protein is active as oligomeric form in darkness and achieves allosteric photo-deactivation upon light, providing intriguing mechanistic insight into the molecular switch for developmental transition.

Mutations in eIF5B Confer Thermosensitive and Pleiotropic Phenotypes via Translation Defects in Arabidopsis thaliana.

The conserved eukaryotic translation initiation factor 5B, eIF5B, is a GTPase that acts late in translation initiation. We found that an Arabidopsis thaliana mutant sensitive to hot temperatures 3 (hot3-1), which behaves as the wild type in the absence of stress but is unable to acclimate to high temperature, carries a missense mutation in the eIF5B1 gene (At1g76810), producing a temperature sensitive protein. A more severe, T-DNA insertion allele (hot3-2) causes pleiotropic developmental phenotypes. Surprisingly, Arabidopsis has three other eIF5B genes that do not substitute for eIF5B1; two of these appear to be in the process of pseudogenization. Polysome profiling and RNA-seq analysis of hot3-1 plants show delayed recovery of polysomes after heat stress and reduced translational efficiency (TE) of a subset of stress protective proteins, demonstrating the critical role of translational control early in heat acclimation. Plants carrying the severe hot3-2 allele show decreased TE of auxin-regulated, ribosome-related, and electron transport genes, even under optimal growth conditions. The hot3-2 data suggest that disrupting specific eIF5B interactions on the ribosome can, directly or indirectly, differentially affect translation. Thus, modulating eIF5B interactions could be another mechanism of gene-specific translational control.

Molecular and Functional Characterization of Wheat ARGOS Genes Influencing Plant Growth and Stress Tolerance.

Auxin Regulated Gene involved in Organ Size (ARGOS) is significantly and positively associated with organ size and is involved in abiotic stress responses in plants. However, no studies on wheat ARGOS genes have been reported to date. In the present study, three TaARGOS homoeologous genes were isolated and located on chromosomes 4A, 4B, and 4D of bread wheat, all of which are highly conserved in wheat and its wild relatives. Comparisons of gene expression in different tissues demonstrated that the TaARGOSs were mainly expressed in the stem. Furthermore, the TaARGOS transcripts were significantly induced by drought, salinity, and various phytohormones. Transient expression of the TaARGOS-D protein in wheat protoplasts showed that TaARGOS-D localized to the endoplasmic reticulum. Moreover, overexpression of TaARGOS-D in Arabidopsis resulted in an enhanced germination rate, larger rosette diameter, increased rosette leaf area, and higher silique number than in wild-type (WT) plants. The roles of TaARGOS-D in the control of plant growth were further studied via RNA-seq, and it was found that 105 genes were differentially expressed; most of these genes were involved in 'developmental processes.' Interestingly, we also found that overexpression of TaARGOS-D in Arabidopsis improved drought and salinity tolerance and insensitivity to ABA relative to that in WT plants. Taken together, these results demonstrate that the TaARGOSs are involved in seed germination, seedling growth, and abiotic stress tolerance.

Molecular epidemiology of noroviruses associated with sporadic gastroenteritis in Guangzhou, China, 2013-2015.

Norovirus diarrhea is a great threat to public health worldwide. To characterize the prevalence of circulating noroviruses associated with sporadic gastroenteritis cases in Guangzhou, 215 stool specimens were collected during two consecutive cold seasons in 2013-2015. Noroviruses were detected in 25 (11.63 %) samples, and GII.4 (6/9) and GII.17 (10/16) were identified as the most predominant variants of each of those seasons. The remaining strains belonged to the genotypes GII.P12/GII.3, GII.2, and GI.Pb/GI.6. The phylogenetic relationships of the GII.17 strains were analyzed based on their capsid protein sequences. This study suggests a significant shift of predominant variants associated with sporadic gastroenteritis in Guangzhou.

Comparative genome analysis of a norovirus GII.4 strain GZ2013-L10 isolated from South China.

In this study, the genome sequence of a norovirus GII.4 strain isolated from South China was comparatively analyzed. The RNA genome of the strain GZ2013-L10 was composed of 7513 nucleotides. Phylogenetic analyses based on three ORFs confirmed its genotype as GII.Pe/GII.4-2012. Compared with other 22 genomes of the same variant, nine distinct nucleotide substitutions were found in the new genome, which resulted in three amino acid changes. All 138 capsid protein VP1 sequences of GII.4-2012 variants were also collected, and multiple alignments revealed 35 variable codons. Evolutionary analyses of GII.4-2012 variants were performed against previous pandemic GII.4 variants, and 2 distinctive changes were identified on epitopes A and E (E368, T413), which resulted in an obvious variation of their solvent-accessible surface areas. Therefore, the genome of GZ2013-L10 was extensively characterized, and new emerging variations on viral epitopes were predicted to contribute to NoV persistence in humans.

Development of a sensitive method for directly sequencing GII.4 norovirus genome.

A new strategy was proposed for amplifying and sequencing GII.4 norovirus genomes directly. A set of primer pairs was rationally designed, which amplified 6 overlapping fragments encompassing the whole genome. The sensitivity of new primers was comparable to that of detection primers, and 10 viral genome sequences were successfully obtained.