Search Results (234)

Human immunodeficiency virus type 1 exhibits extensive genetic diversity, which has important implications for molecular epidemiology, recombinant-pattern assessment, and antiretroviral resistance surveillance. In Mexico, HIV-1 molecular surveillance has historically relied mainly on partial pol gene sequencing, limiting the ability to compare lineage assignments across gag, pol, and env regions. We analyzed plasma samples from 40 treatment-naïve adults receiving care at a tertiary-care hospital in Mexico using a commercial amplicon-based multiregion HIV-1 genomic sequencing workflow. DeepChek^® was used as the primary workflow for read processing, mutation calling, region-level subtype assignment, and antiretroviral resistance interpretation. Resistance interpretation was restricted to antiretroviral target regions with sufficient coverage, mainly reverse transcriptase, protease, integrase, and capsid, when available. Drug resistance mutations were identified in 6/40 participants (15.0%) when mutation-level resistance findings in RT, PR, and IN were considered; one additional sample showed a capsid inhibitor-nonsusceptible NGS call. NNRTI-associated findings were identified in 2/40 patients (5.0%), whereas NRTI- and PI-associated findings were identified in 1/40 patients (2.5%). Accessory or secondary INSTI-associated substitutions were detected in 2/40 patients (5.0%). Region-level subtype analysis revealed frequent discordant assignments across amplified segments, which is consistent with complex mosaic profiles; however, these findings are interpreted as region-level subtypes and recombinant-pattern assignments rather than continuous whole-genome recombination maps. One sample had insufficient RT/PROT/INT coverage for drug resistance interpretation in the complete DeepChek report and was retained only for regions meeting quality thresholds. These findings support the value of multiregion HIV-1 sequencing for local molecular surveillance while emphasizing the need for transparent region-level coverage reporting, cautious interpretation of recombinant-pattern calls, and transparent repository reporting. Full article

Background/Objectives: Determining homologous recombination repair (HRR) status in metastatic castration-resistant prostate cancer (mCRPC) is essential to ensure access to targeted therapies, particularly PARP inhibitors. Yet, variability in testing access and analysis performance exists. This study evaluated feasibility and outcomes of a centralized HRR testing strategy in Spain for prostate cancer patients. Methods: A total of 1412 formalin-fixed paraffin-embedded (FFPE) tumor samples from mCRPC patients from 89 Spanish institutions within a centralized multicenter molecular testing program were analyzed using a standardized 38-gene-based next-generation sequencing (NGS) assay in a central laboratory (HRR OncoKit, Health in Code, Valencia, Spain), which included five clinically relevant HRR genes: BRCA1, BRCA2, CHEK2, ATM, and CDK12. Results: HRR gene pathogenic or likely pathogenic alterations were identified in 18% (CI 95% = 16–20) of the patients, with BRCA2 being the most frequently altered gene (6%), followed by ATM (5%), CDK12 (4%), BRCA1 (2%), and CHEK2 (1%). Eleven percent had variants of uncertain significance. Only 13% of the samples were rejected due to poor DNA quality, low tumor content or sample age exceeding 5 years, and 2% of the samples analyzed failed since the minimum library technical quality score was not achieved. The average turnaround time for results was 18 ± 3 days. Conclusions: Centralized HRR testing in mCRPC patients in Spain was feasible, efficient and reliable, identifying pathogenic alterations in 18% of the cases, similarly to the prevalence described in the literature. This testing approach facilitates precision medicine by improving the detection of actionable HRR alterations. Full article

Background: Point-of-care (POC) glucometers are essential for rapid blood glucose monitoring but are subject to interference and hematocrit variations. This study evaluated the analytical performance of the new Cobas Pulse glucometer against the Accu-Chek Inform II meter in the presence of N-acetylcysteine (NAC, 0.32–2.5 mmol/L), ascorbic acid (0.28–2.84 mmol/L), D-galactose (5.5–27 mmol/L), hemolysis (0.5–5 g/L hemoglobin), icterus (200–1600 μmol/L bilirubin), lipemia (2.5–15 g/L Intralipid), and hematocrit variations (20–60%). Methods: Interference testing followed CLSI EP07 guidelines using three whole blood pools with low (2.0–2.7 mmol/L), medium (4.5–7.4 mmol/L), and high (16.3–23 mmol/L) glucose levels. Interferents were spiked into these whole blood pools. Duplicate glucose levels were measured by 2 Pulse meters and 2 Inform II meters. The results were then assessed using the international standards, e.g., ISO 15197:2017 criteria (±15% or ±0.83 mmol/L). Results: Accu-Chek Inform II showed severe positive interference from galactose (up to 446.3%, p < 0.001), ascorbic acid (up to 98.8%, p = 0.002), and NAC (up to 61.4%, p = 0.001), exceeding ISO limits. Cobas Pulse demonstrated minimal interference (maximum biases: −3.7% for galactose, −4.4% for ascorbic acid, 7.7% for NAC, all p > 0.05). Both meters showed similar hematocrit-dependent bias (positive at 20–30%, negative at 50–60%) and acceptable performance for hemolysis, icterus (≤800 μmol/L), and lipemia. Conclusions: Compared to the Accu-Chek Inform II, the Cobas Pulse demonstrated greater resilience to interferences. Cobas Pulse meets strict accuracy standards (±10% for hospital use) with low interference, which makes it suitable for care of critically ill patients. The Cobas Pulse is more dependable for POCT across various clinical situations, supporting its role in critical care. Full article

Wilms tumor (WT), the most common kidney neoplasm in children, is closely associated with hereditary factors. This study included 134 WT patients (62 males, median age of 7 years, age at diagnosis of 24.9 months) with unilateral (n = 90, 67%) or bilateral WT (n = 44, 33%). Genetic testing was performed using targeted sequencing of 415 genes and multiplex ligation–dependent probe amplification (MLPA). Twenty-five mutations in eight genes were found in 17% (n = 23) of patients: WT1 (n = 10), TRIM28 (n = 4), REST (n = 3), CHEK2 (n = 3), BRCA2 (n = 2), NF1 (n = 1), RAD50 (n = 1), and CDC73 (n = 1). Large deletions of the 11p13 region were revealed in 6% (n = 5) of patients. The 11p15 locus methylation was studied in blood, tumor, and healthy kidney tissue of nine patients suspected of Beckwith–Wiedemann syndrome (BWS) using methylation-sensitive MLPA (MS–MLPA). BWS was diagnosed in 3% (n = 4) of cases (one patient had mosaic disease). Thus, genetic and epigenetic aberrations were identified in 32 WT patients (24%). These patients had a higher frequency of bilateral WT and a higher rate of abnormalities compared to patients without aberrations (56% vs. 25%, p = 0.002; and 86% vs. 25%, p < 0.0001, respectively). The detection of WT hereditary predisposing factors is crucial for treatment strategies and long-term patient surveillance. Full article

Background: Recurrence poses a major challenge in epithelial ovarian cancer (EOC), often occurring despite optimal first-line therapy. Dormant cancer cells are believed to play a key role, yet the mechanisms driving their reactivation remain unclear. This study examined whether exosomes released by normal peritoneal mesothelial cells (PMCs) and fibroblasts (PFBs) undergoing iatrogenic senescence after carboplatin and paclitaxel exposure contribute to EOC recurrence. Methods and Results: Senescent PMCs and PFBs secreted markedly more exosomes, identified by CD9, CD63, and CD81, compared with young cells. Exosomes from both cell types more effectively reactivated dormant EOC cells (pEOCs, A2780, OVCAR-3, SKOV-3) than non-exosomal medium constituents. Importantly, senescent PMC-derived exosomes most strongly reactivated pEOCs and SKOV-3, whereas those from senescent PFBs exerted greater effects on pEOCs, OVCAR-3, and SKOV-3. Kinetic studies of exosome internalization revealed that this process was generally more efficient in the presence of exosomes derived from senescent cells compared with those from young donor cells. Compositional analysis revealed distinct profiles between young and senescent exosomes compared in two variants: young PMCs/senescent PMCs and young PFBs/senescent PFBS. Senescent PMC exosomes displayed reduced miR-210-3p, miR-409-3p, and miR-421, alongside elevated MMP1, MMP3, and VEGF, while senescent PFB exosomes showed increased amphiregulin and osteopontin but lower MMP1, MMP3, TIMP1, bFGF, VEGF, and HGF. Functionally, senescent PMC exosomes enhanced pEOC migration, invasion, and spheroid formation, and induced the expression of CCL11 and ABCB1. Senescent PFB exosomes promoted migration and upregulated CCL11, TGF-β1, BIRC5, and CHEK1. Conclusions: These findings suggest that therapy-induced senescence in peritoneal cells may contribute to EOC recurrence by reactivating dormant tumor cells through exosomal signaling. Full article

The use of postmortem (autopsy) material in fundamental and applied biomedical research significantly facilitates the collection of biomaterial for statistically robust sample cohorts. However, natural adaptive processes to developing cellular stress in the early postmortem period, caused by oxygen and nutrient deprivation, trigger the activation of numerous genes promoting cell survival under stress. Many of these activated pathways are also crucial for tumor cell survival in vivo, as evidenced by various transcriptomic studies. This study aimed to investigate the potential influence of postmortem interval (PMI) duration on gene expression in normal and tumor tissues. Using a model of chemically induced hepatocellular carcinoma in mouse liver, we comparatively analyzed the dynamics of transcript levels for several genes (BRCA1, BRCA2, CHEK1, CHEK2, ATM, CDK12) in paired samples of normal and tumor tissue over a 24-h PMI using RT-qPCR. In normal tissue, gene expression increased significantly, while tumor tissue demonstrated relative transcriptional stability, with no substantial changes in the studied transcript levels. A critical finding was the observed convergence of expression profiles: initial differences between the tissues were completely eliminated by 24 h PMI. This pattern developed despite formally adequate RNA quality (RQN) and the absence of clear signs of progressive autolysis in histology, indicating the insufficiency of standard quality criteria for detecting postmortem changes. These findings collectively underscore the critical importance of minimizing and controlling PMI during the biobanking of oncological samples for reliable transcriptomic research. Full article

Chronic lymphocytic leukemia (CLL) exhibits marked clinical heterogeneity that is closely associated with genomic instability. Although cytogenetic abnormalities are widely used for risk stratification, they do not fully capture the biological complexity of the disease. Telomere dysfunction and alterations in DNA damage response pathways have been implicated in disease progression, but their relationship with cytogenetic risk in CLL remains incompletely characterized. In this study, peripheral blood mononuclear cells (PBMCs) from 48 CLL patients were analyzed. The analyzed PBMC fractions were enriched in leukemic B cells, with an estimated median tumor content above 85–90%. Cytogenetic profiles were obtained by conventional karyotyping following in vitro immunostimulation with DSP30 and interleukin-2 and classified according to ERIC and Döhner criteria. Telomere length was assessed by quantitative PCR, and CHEK1 and CHEK2 expression levels were quantified by RT–qPCR. Molecular parameters were compared across cytogenetic risk groups. Distinct molecular profiles were observed across cytogenetic categories. Favorable-risk CLL cases showed preserved telomere length, low CHEK1 expression, and maintained CHEK2 levels. Intermediate-risk cases, predominantly characterized by trisomy 12, exhibited moderate telomere shortening accompanied by increased CHEK1 expression and partial reduction of CHEK2. High-risk CLL cases, defined by del(11q), del(17p), or complex karyotypes, displayed pronounced telomere shortening, marked CHEK1 upregulation, and strong suppression of CHEK2. Telomere length was inversely correlated with cytogenetic risk (Spearman’s ρ = −0.68, p < 0.0001), and the CHEK1/CHEK2 expression ratio increased progressively with genomic complexity. These findings indicate that telomere length and CHEK1/CHEK2 expression patterns are closely associated with cytogenetic risk in CLL and may provide complementary biological information for risk stratification. Full article

Background: Breast cancer (BC) is one of the most diagnosed malignancies and a leading cause of cancer-related mortality among women worldwide, thereby posing a substantial threat to women’s health worldwide. However, clinically robust diagnostic biomarkers with high sensitivity and specificity, as well as well-validated molecular targets for targeted therapy, remain limited. Methods: BC transcriptomic data from seven GEO datasets and the TCGA-BRCA cohort (n = 1231) were integrated for analysis. After batch-effect correction, candidate genes were screened through DEA, WGCNA, and PPI networks analysis. An ensemble machine learning (ML) framework incorporating 127 algorithmic combinations was constructed, and SHAP analysis was applied to identify hub genes. Further analyses included functional enrichment, immune infiltration, miRNA regulatory network analysis, and SMR analysis. The expression patterns were validated using single-cell transcriptome data. Drug repositioning analysis and AI-assisted virtual screening were performed to prioritize compounds with favorable drug-like properties. The predicted binding modes of candidate compounds with CHEK1 were assessed by molecular docking. Results: Thirty core genes were obtained through differential expression, WGCNA, and PPI screening. Integrated ML (127 algorithms) determined the optimal model (AUC = 0.919), and SHAP identified nine feature genes, among which CHEK1 and KIF23 showed preliminary diagnostic potential across four external cohorts (AUC: 0.625–0.938). Functional enrichment indicated that both are enriched in the cell cycle and p53 pathways, closely associated with BRCA1/ATR; immune infiltration revealed significant correlations with macrophages and CD8⁺ T cells, with hsa-miR-15a-5p and hsa-miR-607 being common upstream regulatory miRNAs. SMR analysis supported a causal relationship between CHEK1 expression and BC genetic susceptibility (p_SMR < 0.05, p_HEIDI > 0.05); single-cell analysis confirms its heterogeneous expression. AI-assisted virtual screening identified 25 A-grade computational candidate compounds from 171 candidates. Molecular docking suggested that Olaparib and LY294002 can form favorable interactions with the CHEK1 active pocket. Conclusions: The study identified CHEK1 as a key diagnostic gene for BC through 127 ML algorithms and SMR causal inference. By combining AI-assisted virtual screening and molecular docking, computational candidate compounds targeting CHEK1 were prioritized. These findings represent hypothesis-generating in silico predictions and require experimental validation before any therapeutic conclusions can be drawn. Full article

Background/Objectives: Contralateral breast cancer (CBC) is a significant concern among breast cancer survivors, particularly in those with moderator-high penetrance germline mutations such as BRCA1, BRCA2, CHEK2, and ATM. While radiotherapy (RT) is a crucial component of breast cancer (BC) treatment, its potential role in increasing CBC risk remains unclear. This systematic review and meta-analysis aim to evaluate the incidence of radiation-induced CBC in germline mutation carriers. Methods: Following PRISMA guidelines, we conducted a comprehensive search in six electronic databases (PubMed, Scopus, Cochrane Library, ProQuest, EBSCO, and Epistemonikos) for studies published fifteen years prior, up to August 2025. We included cohort and case–control studies assessing the association between RT and CBC incidence in germline mutation carriers. A meta-analysis was performed using a random-effects model to estimate cumulative risk (CR) and rate ratios (RR). Results: Seven studies were included. The 5-year cumulative risk (CR) of contralateral breast cancer (CBC) was 0.55 for BRCA1/2, 0.89 for ATM, and 0.80 for CHEK2 carriers. At 10 years, overall CR increased to 0.65, with ATM and CHEK2 remaining high. Rate ratio (RR) analysis showed a significant risk for ATM (2.98), while overall RR indicated more than a two-fold increased CBC risk with radiotherapy (RR = 2.70 common-effect; 2.53 random-effects). Conclusions: Radiotherapy significantly increases contralateral breast cancer risk, particularly in ATM and CHEK2 carriers, emphasizing the importance of personalized genetic risk stratification in treatment decisions. Full article

Background/Objectives: Influenza, RSV, and SARS-CoV-2 co-circulate and evolve under immune and therapeutic pressures, complicating decision-making for both vaccine formulation and antiviral use. Fragmented, pathogen-specific sequencing approaches limit cross-virus comparability. Methods: We applied a standardized, multiplexed, amplicon-based next-generation sequencing (NGS) workflow to 34 diagnostic specimens (Ct < 35) positive for influenza A/B, RSV-A/B, or SARS-CoV-2. Sequencing libraries were generated and run on an Illumina MiSeq platform (2 × 250 bp). Although the wet-lab workflow is standardized across pathogens, consensus generation and annotation utilized two different analysis environments: Geneious Prime for influenza and MicrobioChek for RSV and SARS-CoV-2. Quality metrics included genome breadth and depth of coverage. Results: Near-complete genomes (mean coverage ≥98%) were recovered for all samples. Influenza A(H1N1)pdm09 sequences clustered in clade 6B.1A; A(H3N2) clustered in subclade 3C.2a1b.2a.2; and influenza B belonged to the Victoria lineage V1A.3a.2. RSV sequences were assigned to Nextclade clades A.D.5.1, A.D.1.10, A.D.2.1, and A.D.3 (RSV-A) and to B.D.4.1.3 and B.D.E.1 (RSV-B), consistent with the ON1 (RSV-A) and BA (RSV-B) genotypes prevalent in recent seasons. Clinically relevant mutations included changes in the influenza HA site and neuraminidase substitutions, RSV F-protein polymorphisms, and spike protein substitutions associated with recent Omicron sublineages (L455F/S, F456L) in SARS-CoV-2. Conclusions: A unified amplicon–NGS approach yields harmonized genomic data across respiratory viruses, enabling timely detection of antigenic drift and resistance markers while supporting integrated, cross-pathogen surveillance. Full article

Accurate perioperative glucose monitoring is essential in dogs undergoing general anesthesia, yet most validation studies of handheld glucometers have been performed under stable outpatient conditions. This prospective clinical validation study evaluated the analytical agreement, diagnostic performance, and ISO 15197 compliance of a human-calibrated (Accu-Chek) and a veterinary-specific (Centrivet GK) handheld glucometer compared with a laboratory spectrophotometric reference method in 34 anesthetized dogs (99 paired measurements per device). Linear mixed-effects modeling demonstrated significant method effects (p < 0.001), with the veterinary-specific device overestimating glucose concentrations relative to the reference method (β = 20.79 mg/dL; 95% CI: 8.08–33.50; p = 0.001), whereas the human-calibrated device did not differ significantly (β = 7.18 mg/dL; 95% CI: −5.53–19.89; p = 0.267). Bland–Altman analysis showed mean bias of 4.44 mg/dL (95% CI: 0.73–8.16) for the human-calibrated device and 22.72 mg/dL (95% CI: 18.22–27.21) for the veterinary-specific device. Passing–Bablok regression identified proportional bias only for the veterinary-specific device (slope 1.19; 95% CI: 1.01–1.34). ISO compliance was 69.7% and 39.4%, respectively. For hyperglycemia detection, AUC values were 0.9566 (95% CI: 0.8955–1.0000) and 0.9757 (95% CI: 0.9479–1.0000); for hypoglycemia, 0.8567 (95% CI: 0.7557–0.9578) and 0.7376 (95% CI: 0.6056–0.8697). In anesthetized dogs, the human-calibrated device demonstrated superior analytical robustness, whereas the veterinary-specific device showed greater bias and variability. Full article

Background/Objectives: Pain is a common symptom for hospitalised older adults. Pain is not always adequately assessed, which can lead to inadequate pain management and adverse patient outcomes. Thus, new technology-driven pain assessment tools have been developed; however, little is known about patients’ and families’ experiences of nurses using them in acute care. This study aimed to explore the perspectives of older adult inpatients and their families’ regarding nurses’ use of the digital technology-driven pain assessment application PainChek^® Universal. Methods: A survey was undertaken as part of a stakeholder evaluation of a randomised control trial exploring the effectiveness of nurse-led volunteer support and technology-driven pain assessment in improving the outcomes of hospitalised older adults. The PainChek^® Universal application was implemented on two medical wards of an acute private hospital in Western Australia as part of a larger single-centre, prospective, non-blinded, cluster-randomised control trial. This stakeholder evaluation invited older adult inpatients and their family members to participate in a survey about nurses’ use of the PainChek^® Universal application for pain assessment. Results: A total of 96 inpatients and 27 family members completed the survey. Thirteen patients and nine family members provided additional feedback. Over 90% of patients and family members agreed that the use of the PainChek^® Universal application was a positive addition to pain assessments, rendered no concerns, and helped nurses complete pain assessments. A total of 84% of patients and 87% of family members felt PainChek^® Universal provided a more accurate pain assessment. Survey feedback related to PainChek^® Universal application use, integration of technology, and need for further education. Conclusions: The findings suggest that older adults and their families recognised the benefits of nurses using a digital application for pain assessments. Technology integration in healthcare must be accompanied by patient and family education. Full article

Infectious bronchitis virus (IBV) is a highly infectious disease of chickens posing a major threat to poultry production worldwide. Due to genetic and antigenic variability, it is difficult to establish one universal protocol for vaccination against IB. The protectotype vaccination strategy, combining antigenically distinct vaccine strains, offers broader cross-protection than homologous IBV vaccination approaches. This field study aimed to establish serological baseline values in broiler chickens in a protectotype vaccination program using Avishield IB H-120 and Avishield IB GI-13, and to evaluate the program’s efficacy under field conditions on commercial farms where heterologous IBV genotypes were detected. The study was conducted on 25 broiler farms in 2023–2024. Day-old chicks were vaccinated via coarse spray. Blood samples were taken from them at six weeks of age and analyzed using IDEXX and BioChek ELISAs, and cecal tonsils were tested by real-time RT-PCR. Production performance was assessed using the European production efficiency factor (EPEF), with a cut-off value of 360. No IB-related clinical signs were reported, and the mean EPEF of the included farms was 408.7. The detected IBV genotypes were Mass + 793B, 793B, and 793B + VAR2. The presence of heterologous VAR2 did not negatively impact flock health or performance. Preliminary serological baseline values for vaccinated, non-VAR2 farms were established at 891–1332 (in chicks tested with the IDEXX ELISA) and 4193–5204 (in those tested with the BioChek assay). The H-120 + GI-13 program demonstrated high field efficacy, reliable serological profiles, and cross-protection in the presence of heterologous IBV. Continuous monitoring of IBV genotypes remains essential. Full article

Glioblastoma is a highly invasive primary brain tumor with a poor prognosis, highlighting the need for new therapeutic strategies. Toxins derived from Macrodactyla doreensis have attracted attention for their potential anticancer activity. This study evaluated the anticancer and cytotoxic effects of M. doreensis crude venom on two commonly used glioblastoma cell lines (U251 and LN229), which mirror the phenotype of primary tumors. Cell viability and proliferation were assessed using the CCK-8 assay and colony formation assay, while cell migration and invasion capabilities were detected via wound healing assay and Transwell assay. Annexin V/PI staining and PI-based cell cycle analysis indicated that the crude venom significantly induced cell apoptosis and caused S-phase arrest. Proteomic analysis combined with GO and KEGG enrichment analyses as well as bioinformatics approaches showed that M. doreensis crude venom inhibits glioblastoma cell proliferation by downregulating the expression of CDK2, RRM2, and CHEK1, thereby hindering cell cycle progression and regulating the p53 signaling pathway. Notably, the downregulation of these key glioblastoma-related target genes was validated by qPCR. In addition, network pharmacology analysis indicated that several peptide families present in the sea anemone crude venom, including ShK peptides, inhibitor cystine knot (ICK) peptides, and EGF-like peptides, exhibit notable antitumor potential. Combined with AlphaFold2-based structural modeling and molecular docking, these analyses further elucidated the potential molecular mechanisms underlying their interactions with key targets, such as MD-381 with RRM2, MD-322 with CDK2, and MD-429 with CHEK1. Collectively, these findings highlight the therapeutic potential of M. doreensis crude venom and lay a foundation for the subsequent isolation of novel peptides and their further development in glioblastoma treatment. Full article

Human preimplantation embryo arrest (PREMBA) represents a significant clinical hurdle in assisted reproductive technology (ART), in which approximately 10% of in vitro fertilized (IVF) embryos arrest at the cleavage stages. Whole-exome sequencing (WES) studies have discovered numerous genetic mutations associated with preimplantation embryo arrest. These mutations often disrupt critical biological milestones such as maternal mRNA clearance (BTG4, ZFP36L2, ZAR1), subcortical maternal complex (TLE6, PADI6, OOEP, NLRP2, NLRP5, NLRP7, KHDC3L), DNA double-strand break formation and homologous recombination (REC114, TOP6BL, MEI1, MEI4, TRIP13), spindle assembly (TUBB8 and TUBA4A) and cell cycle and checkpoints (FBXO43, MOS, CHEK1, TRIP13, CDC20), as well as nuclear transport and translational regulation (KPNA7, DDOST). However, the cause of most clinical cases remains genetically unexplained. Studies investigating these unexplained arrests have uncovered widespread multi-omics abnormalities, including transcriptional arrest, DNA hypermethylation, higher chromatin accessibility, aberrant histone modification, chromosomal aneuploidy and senescent-like states. This review provides a comprehensive overview of the molecular mechanisms underlying PREMBA, categorized into those that are attributable to known genetic mutations and those with unexplained reasons. Full article