GATM; c.965G>C p.(Arg322Pro) segregating dominantly in the mother and daughter. We validated our finding with molecular dynamics simulations and demonstrated a dynamic signature that differentiates our variant and two previously identified pathogenic variants in GATM from wildtype. Genetic testing has uncovered a novel pathogenic variant that predicts progression to end stage kidney failure and has important implications for family planning and cascade testing. We recommend that GATM is screened for in children presenting with RFS, in addition to adults, particularly with kidney failure, who may have had previous negative gene testing.Renal Fanconi syndrome (RFS) is a generalised disorder of the proximal convoluted tubule. Many genes have been associated with RFS including those that cause systemic disorders such as cystinosis, as well as isolated RFS. We discuss the case of a 10‐year‐old female who presented with leg pain and raised creatinine on a screening blood test. Her mother has RFS and required a kidney transplant in her thirties. Further investigations confirmed RFS in the daughter. Exome sequencing was performed on the affected mother, child, and unaffected father. We identified a novel variant in  GATM, c.965G>C p.(Arg322Pro). Molecular dynamic simulation confirmed that the abnormal protein product would tend to form multimers likely to disrupt mitochondrial function.Whole exome sequencing of a mother and daughter with isolated Fanconi syndrome revealed a novel variant of  To date, several genes have been associated with RFS, of which some cause systemic disorders with RFS and others cause isolated RFS with or without kidney failure Table .SLC34A1,EHHADH,NDUFAF6.GATM as the causal gene in the 15q15.3 locus. GATM was previously missed by Lichter‐Konecki et al. as they had erroneously excluded flanking markers which excluded GATM.The genetic aetiology of isolated RFS is still being elucidated. In 2001, Lichter‐Konecki et al.22. She was referred to Southampton's paediatric nephrology service. Her mother presented at a similar age with leg pain, polyuria and polydipsia. Aged 14, she was found to have rickets and was referred for further investigation. These confirmed RFS with a plasma creatinine concentration of 53 μmol/L. Aged 17 her plasma creatinine concentration had risen to 104 μmol/L and her GFR measured by EDTA plasma disappearance was 57 ml/min/1.73 m2. She progressed to end‐stage kidney disease and had a live, unrelated kidney transplant in 2020 (aged 38). Neither of the maternal grandparents had any features to suggest RFS and neither developed chronic kidney disease. A maternal uncle had completely normal kidney function.A 10‐year‐old white female presented with bilateral knee pain. Screening blood tests showed a raised plasma creatinine with an eGFR of 65 ml/min/1.73 mBiochemical investigations of the presenting daughter revealed low plasma concentrations of bicarbonate (13 mmol/L), potassium (3.3 mmol/L), and inorganic phosphate (0.89 mmol/L). Urinary protein/creatinine ratio was elevated (124 mg/mmol) and her lactate was raised at 2.5 mmol/L. Alkaline phosphatase was also raised (540 U/L). Plasma glucose, sodium, magnesium, and parathyroid hormone were within normal ranges. She had heavy generalised aminoaciduria and raised urinary retinol binding protein/creatinine ratio . She had intermittent glucosuria. A lower limb X‐ray revealed widening of the distal femoral and proximal tibial growth plates suggestive of rickets. Upper limb X‐rays were unremarkable. Abdominal and kidney ultrasound scan revealed bilateral medullary nephrocalcinosis. There were no other features to suggest a mitochondrial disorder. Normal white blood cell cystine (0.05 nmol ½cystine per mg protein) excluded cystinosis. Fructose intolerance was excluded due to absence of jaundice or vomiting. Galactosaemia was ruled out due to absence of cataracts and hepatosplenomegaly. Absence of persistent glycosuria, hypoglycaemia, and hepatomegaly excluded Fanconi–Bickel syndrome. Therefore, the patient was diagnosed with RFS of unknown aetiology. However, with her mother's progression to end‐stage kidney disease and evidence of an impaired eGFR, there was high suspicion for an autosomal dominant genetic cause with a similar disease trajectory.fastq to vcf using an automated joint‐calling pipeline, aligned to GRCh38. Quality control ensured only high‐quality variants remained for downstream analysis. Variants were annotated with Ensembl VEP v103 and the resultant joint‐called vcf was uploaded to a local installation of seqr (https://github.com/broadinstitute/seqr) for data visualisation, analysis, filtering, and reporting.Trio exome sequencing  was performed to investigate a molecular cause. Library capture was performed using Agilent SureSelect Human All Exon V6 and DNA were sequenced by Novogene. Data were processed from To filter our data, we applied the Renal Tubulopathies V 2.30 PanelApp gene panel and restricted our analysis to: a dominant inheritance pattern; all exonic variants excluding synonymous; allele frequency <0.001 from gnomAD v2.1.1A total of 96 894, 96 370, and 96 830 variants were called in the daughter, mother, and unaffected father respectively. Average read depth was 53, 49, and 49 respectively. Post‐filtering, two variants remained Table .KCNJ1 was considered an incidental finding and not causal for RFS. Pathogenic variants in KCNJ1 are autosomal recessiveGATM: c.965G>C p.(Arg322Pro), had a genotype quality of 99 in all sequenced individuals and a depth of 74 in the proband, 74 in the mother, and 37 in the father. The variant was confirmed in the proband and mother by Sanger sequencing.The variant in 2.1GATM variant. The classical dimer B2–B2 interaction also required for multimerization was not included in simulations as we expect this interaction to be invariant across both pathogenic and non‐pathogenic states due to its existence in the native form, and our prior simulations of B2–B2 dimers did not indicate any notable differences between variants. Further simulation details are provided in the GATM variants on large scale motions observed in the trajectories  variant, we performed conventional molecular dynamics simulations on wildtype (WT), p.(Arg322Pro) (our variant), and two pathogenic variants, p.(Thr336Ala) and p.(Pro320Ser), as reported by Reichold et al.s Figure . On visus Figure . Relativs Figure  and S1.3GATM encodes a proximal tubular nuclear‐encoded mitochondrial enzyme, glycine amidinotransferase, which catalyses the transfer of a guanidino group from l‐arginine to glycine, resulting in guanidinoacetic acid, an immediate precursor to creatine.GATM expression is limited to the highly energy dependent proximal tubular cells, which have abundant mitochondria to support the oxygen‐dependent generation of ATP.GATM cause a rare, congenital neurological disorder without kidney dysfunction.GATM affecting highly conserved residues, segregating in an autosomal dominant pattern. The four causal variants, p.(Pro320Ser); p.(Thr336Ala); p.(Thr336Ile); and p.(Pro341Leu), were fully penetrant and clustered on conserved proline and threonine residues representing <5% of the protein. In silico modelling suggested the variants could adversely affect protein folding and cause GATM to form longitudinal multimers. Biopsy samples from affected patients demonstrated fibrosis and extremely large, filament‐filled mitochondria within proximal tubule cells. These were confirmed to contain GATM using immunogold staining.GATM had no impact on mitochondria, but all mutants caused structural deformity to mitochondria consistent with the findings on patient biopsies. LLC‐PK1 cells  transfected with the p.(Thr336Ala) variant had no effect on enzymatic activity and oxidative phosphorylation, but instead caused dimeric GATM to form multimers, resulting in fibrillary aggregation within the mitochondria consistent with the in silico modelling.NLRP3, a component of the inflammasome, elevated pro‐inflammatory cytokine interleukin‐18, increased profibrotic factors and increased cell death. Furthermore, mutant GATM showed impaired protein degradation with an increased half‐life. Of note, mice haploinsufficient for GATM had no significant kidney phenotype, suggesting the four variants cause disease through a dominant negative mechanism. These mice did however display neurological phenotypes consistent with biallelic loss‐of‐function variants in GATM.GATM expression and 58% reduced protein levels. Therefore, exogenous creatine may suppress endogenous production of mutant GATM and reduce production of abnormal protein aggregates.In 2018, Reichold et al.GATM, segregating in an affected mother‐daughter pair with idiopathic RFS. This variant is two amino acids downstream of a previously described pathogenic variant, p.(Pro320Ser), in GATM causing RFS with progression to kidney failure  variant, NM_001482.3: c.965G>C p.(Arg322Pro), in GATM multimerization as hypothesised by Reichold et al.The p.Arg322 amino acid is extremely conserved across species, and in silico predictors suggest high pathogenicity with a CADD score of 32. To validate pathogenicity of our novel variant we performed conventional molecular dynamics simulations. We tested WT, our variant p.(Arg332Pro), and two previously identified pathogenic variants, p.(Thr336Ala) and p.(Pro320Ser). The two previously reported pathogenic variants by Reichold et al. and our novel variant behaved differently to wildtype. Disease‐associated variants adopted a new ‘close‐contact’ global minimum at ~3.8 Å, whilst WT uniquely retained a ‘distant’ global minimum at ~12.4 Å. Close contacts of residues 320 on both homodimers may be a direct or precursor event to formation of the B4 interaction surface capable of pathogenic The phenotype of our female proband and her affected mother are consistent with the phenotypes reported by Reichold et al.GATM  is included on three gene panels in PanelAppGATM.Currently, 4GATM progress to end stage kidney disease, and therefore we have uncovered a diagnosis that has serious clinical consequences for our patient and will warrant genetic counselling and cascade testing. Early functional work in rats suggests that creatine may be able to significantly reduce expression of the GATM protein and this warrants further investigation.All patients with known pathogenic variants in GATM variants should be routinely tested for in cases of idiopathic RFS even in the absence of renal failure which develops after initial presentation. We further recommend that adult patients showing signs of RFS and chronic kidney disease should also be screened for GATM variants.This case highlights how genetic sequencing and complementary molecular dynamics simulations can identify disease pathogenesis and inform patient care and prognosis. Eleanor G. Seaby processed data, performed data analysis, and wrote the first draft of the manuscript. Steven Turner, David J. Bunyan, and Jonathan Essex performed data analysis. Fariba Seyed‐Rezai assisted with data processing. Sarah Ennis, Jonathan Essex, and Rodney D. Gilbert supervised the project. All authors agreed the final manuscript.Eleanor G. Seaby is supported by the Gerald Kerkut Charitable Trust and the University of Southampton's Presidential Scholarship.The authors declare no conflicts of interest.https://publons.com/publon/10.1111/cge.14235.The peer review history for this article is available at All participants were recruited to the ‘Use of NGS technologies for resolving clinical phenotypes’ study. This study was approved by Leeds East Research Ethics Committee  on the 17/05/17. Written consent was obtained from the parents of the index patient for genomic testing and from the mother for publication after she had seen the manuscript.APPENDIX S1. Supporting InformationClick here for additional data file.FIGURE S1. Molecular dynamics results of GATM B4‐B4 dimer mutantsFree‐energy values for all T336A (a), P320S (b), and R322P_P322R (c) mutants across residue 320 CB atom distances, each replica plotted separately. (def) Free‐energy values for all mutants and WT across top three principal components generated from backbone Cα atom trajectories, averaged across all three replicasClick here for additional data file.TABLE S1. Genetic causes of renal Fanconi syndromeClick here for additional data file.


Accumulating evidences indicate regional gray matter (GM) morphology atrophy in spinocerebellar ataxia type 3 (SCA3); however, whether large‐scale morphological brain networks (MBNs) undergo widespread reorganization in these patients remains unclear.To investigate the topological organization of large‐scale individual‐based MBNs in SCA3 patients.The individual‐based MBNs were constructed based on the inter‐regional morphological similarity of GM regions. Graph theoretical analysis was taken to assess GM structural connectivity in 76 symptomatic SCA3, 24 pre‐symptomatic SCA3, and 54 healthy normal controls (NCs). Topological parameters of the resulting graphs and network‐based statistics analysis were compared among symptomatic SCA3, pre‐symptomatic SCA3, and NCs groups. The inner association between network properties and clinical variables was further analyzed.Cp, lower Eloc, and Eglob . Regarding nodal properties, symptomatic SCA3 exhibited significantly decreased nodal profiles in the central executive network (CEN)‐related left inferior frontal gyrus, limbic regions involving the bilateral amygdala, left hippocampus, and bilateral pallidum, thalamus; and increased nodal degree, efficiency in bilateral caudate . Meanwhile, clinical variables were correlated with altered nodal profiles (pFDR ≤0.029). SCA3‐related subnetwork was closely interrelated with dorsolateral cortico‐striatal circuitry extending to orbitofrontal‐striatal circuits and dorsal visual systems .Compared to NCs and pre‐symptomatic SCA3 patients, symptomatic SCA3 indicated significantly decreased integration and segregation, a shift to “weaker small‐worldness”, characterized by decreased Symptomatic SCA3 patients undergo an extensive and significant reorganization in large‐scale individual‐based MBNs, probably due to disrupted prefrontal cortico‐striato‐thalamo‐cortical loops, limbic‐striatum circuitry, and enhanced connectivity in the neostriatum. This study highlights the crucial role of abnormal morphological connectivity alterations beyond the pattern of brain atrophy, which might pave the way for therapeutic development in the future. Symptomatic SCA3 patients undergo an extensive and significant reorganization in large‐scale individual‐based morphological brain networks, probably due to disrupted prefrontal cortico‐striato‐thalamo‐cortical loop, limbic‐striatum circuitry, and enhanced connectivity in the neostriatum. A cortical feature‐based structural connectivity network can locate atrophied cortical regions and indicate how their connectivity and functions may change. To date, altered GM organization has been reported in SCA3.n = 24) and symptomatic SCA3 (n = 76). We further examine whether the clinical scale for the assessment and rating of ataxia (SARA) scores and disease duration of SCA3 are associated with significant brain GM network properties.Considering distinct infratentorial cerebellar loss/atrophy affecting the organization of whole brain morphological networks, only supratentorial cerebral GM was taken into constructing the networks in this study. Thus, we compute individual‐based GM networks based on the inter‐regional morphological similarities of GM22.1https://clinicaltrials.gov/ (Identifier: ChiCTR2100045857). Written informed consent was obtained from all participants.This study protocol was approved by the ethics committees of our institution and registered at ATXN3 mutation carriers were prospectively recruited. The clinical disease severity was assessed via the scale for the assessment and rating of ataxia (SARA) for each patient.n = 2) and significant registration errors (n = 4)  Figure .The inclusion criteria of SCA3: (1) the molecular genetic testing has proved all the patient's diagnosis; (2) age >18 years; (3) right‐handedness. The exclusion criteria of each participant were as follows: (1) combined with other mental disorders, personality disorders, or psychotropic drug dependence; (2) challenging to cooperate during MRI examination, and the image quality is too poor for image analysis; (3) a history of other brains organic and metabolic diseases; (4) pregnant and lactating women; and (5) other MRI contraindications.2.22, and data matrix = 384 × 384, acquisition time 4 min 21 s.All participants underwent sagittal three‐dimensional T1 imaging with a 3.0‐T MR imaging system . Images were acquired using the Magnetization Prepared Rapid Gradient Echo (MPRAGE) sequence with the following parameters: repetition time (TR) = 1750 ms, echo time (TE) = 2.8 ms, 256 sagittal slices with slice thickness = 0.7 mm with no slice gap, a field of view (FOV) = 260 mm × 260 mmhttp://www.neuro.uni‐jena.de/cat/) within the SPM12 environment (http://www.fil.ion.ucl.ac.uk/spm/software/spm12/) running under MATLAB R2019b (MathWorks). The preprocessing steps involved spatial normalization of the MNI space and segmentation. Modulated GM images were resliced to a 2‐mm isotropic voxel size and spatially smoothed using a 3D Gaussian kernel with an FWHM of 6 mm.Automated segmentation of the whole brain based on 3D T1‐weighted images was processed with the CAT12 toolbox  atlas was applied2.4http://www.nitrc.org/projects/gretna/)S < 0.40 with an interval of 0.05. For the brain networks at each sparsity level, the topologic profiles at both global and nodal levels were calculated. Global network profiles included the clustering coefficient (Cp), characteristic path length (Lp), normalized clustering coefficient (γ), normalized characteristic path length (λ), small‐world parameters (σ), global efficiency (Eglob), local efficiency (Eloc), and nodal network topological profiles including nodal efficiency (Ei), nodal degree (Di), and nodal betweenness (Bi). Considering the network sparsity‐dependent network characteristics, the area under the curve (AUC) of each global profile  and nodal profile  across a range of interested densities were calculated as the summarized scalar for each measure, denoted as Eglobauc, Elocalauc, CPauc, LPauc, γauc, λauc, σauc, Biauc, Eiauc, Diauc, respectively. Finally, BrainNet Viewer toolbox (https://www.nitrc.org/projects/bnv/) was used to present between‐group differences in nodal profiles.GRETNA toolbox (2.5https://www.nitrc.org/projects/nbs/) was used to identify the differences in pairwise connectivity of nodal characteristics between SCA3 and NCs. First, nodes that exhibited significant between‐group differences in at least one of the three nodal centralities  were chosen. Then, a subnetwork connection matrix for each participant was created. Finally, a set of suprathreshold links between connected components were identified using NBS metrics  with the nonparametric permutation approach (5000 permutations).The network‐based statistics (NBS) method  was used to perform statistical analysis. The Shapiro–Wilk test was used to evaluate the normality of the data for continuous variables. Qualitative variables were compared by Chi‐squared tests, and quantitative variables were compared using two‐tailed independent‐sample pFDR < 0.05).Between‐group comparisons of the AUC of each network metric were evaluated with analysis of covariance (ANCOVA) and post hoc analyses with diagnosis as fixed factors, age, and gender added to the model as covariates, respectively. The Benjamini–Hochberg false discovery rate (BHFDR) correction was performed for multiple comparisons. Then, partial correlation analysis was used to examine relationships between significant network metrics and the disease symptom severity (reflected by SARA scores), as well as the disease duration, controlling for age and gender as confounding variables . Compared to NCs, Pre‐SCA3 presented at a younger age (p < 0.001). Detailed demographic and clinical characteristics are summarized in Table Finally, 100 SCA3  were enrolled, including 76 Sym‐SCA3  and 24 pre‐SCA3 . The Sym‐SCA3 group was older than the Pre‐SCA3 group  and Lp values , which showed typical features of small‐world architecture in GM morphological networks. Significant differences in Eglob , Eloc , Lp , Cp , γ , and λ  were observed among the Sym‐SCA3, Pre‐SCA3, and NCs groups. However, no significant difference was observed in λ  among group comparisons.In the defined threshold range, Sym‐SCA3, Pre‐SCA3, and NCs exhibited normalized Eglob (p < 0.001), Eloc (p < 0.001), Lp (p = 0.001), Cp (p < 0.001), γ (p < 0.001), and λ (p < 0.001) values. Meanwhile, compared to Pre‐SCA3, Sym‐SCA3 exhibited significantly decreased Eglob (p = 0.005), Eloc (p < 0.001), Cp (p = 0.003), and increased Lp (p = 0.006) values , nine were in the subcortical cortex, six were in the frontal/prefrontal cortex, five were in the temporal cortex, one was in the parietal cortex, and one was in the occipital cortex , limbic regions , and bilateral thalamus  and nodal degree of the left amygdala , and negatively correlated with nodal efficiency of right caudate . Besides, in Sym‐SCA3 patients, disease duration was positively correlated with nodal efficiency of the left amygdala  and nodal degree of the left amygdala , and negatively correlated with nodal efficiency of left IFGoperc . No significant correlations were found between clinical variables and any other global or nodal metrics (pFDR > 0.05).The SARA scores of Sym‐SCA3 patients were positively correlated with nodal efficiency of the left amygdala 4.1Cp, lower Eloc, and Eglob in Sym‐SCA3 indicated decreased integration and segregation of structural brain networks,Cp, Eglob, and increased Lp  in SCA3. However, a functional connectome study indicated a more regular brain network with higher Cp, Eloc, and modularity in Sym‐SCA3.The shorter 4.2Our work confirms and highlights the disrupted prefrontal cortico‐striato‐thalamo‐cortical loops associated with Sym‐SCA3, which were previously assumedAnother vital circuitry involved with SCA3 was the limbic system. The limbic systemThese findings also extend beyond brain atrophy patterns related to Sym‐SCA3.4.3Decreased nodal efficiency and degree of the left amygdala were positively correlated with SARA scores and disease course. Amygdala is a vital part of the limbic systemIncreased nodal efficiency of the right caudate was negatively corrected with symptom severity in SCA3, which highlights the compensatory and supportive interactions in neostriatum (caudate/putamen) in Sym‐SCA3. Besides, decreased nodal efficiency of CEN‐related left IFGoperc was negatively corrected with the disease course. Within the progress of the disease, the connectivity involving CEN is persistently damaged, emphasizing the importance of early treatment intervention for SCA3.4.4Eglob and Cp, as well as significantly altered nodal properties in the prefrontal, limbic, striatum/thalamus was similarly observed using the HOA‐112 atlas in Sym‐SCA3 than in NCs. Meanwhile, Sym‐SCA3 showed decreased nodal degree and efficiency in the bilateral thalamus and amygdala according to both the AAL‐90 and HOA‐112 atlas, which uncovered the early involvement of the bilateral thalamus and amygdala. In addition, the SCA3‐related subnetwork was similar to those obtained from the AAL‐90 atlas, implying the robustness of these findings.Validation analyses showed that significantly decreased 4.5The current study has two limitations. First, longitudinal studies are needed to examine neurodegenerative alterations from the pre‐SCA3 to Sym‐SCA3 stage to evaluate the impact of the emergence and course of brain atrophy abnormalities on large‐scale brain network organization. Second, due to the distinct infratentorial cerebellar loss/atrophy would affect the organization of whole brain morphological networks, we did not include the infratentorial brain structures in the analysis, which might further limit the exploration of their roles of network alterations in SCA3. Further study with sophisticated network construction methods should be carried out here.5In summary, our findings suggest that Sym‐SCA3 patients undergo an extensive and significant reorganization in MBNs, probably due to disrupted prefrontal cortico‐striato‐thalamo‐cortical loops, limbic‐striatum circuitry, and enhanced connectivity in the neostriatum. Detailed knowledge of large‐scale network reorganization has the potential to help researchers better understand the pathophysiological mechanisms of SCA3 and facilitate the development of therapeutic strategies. In particular, therapeutic modulation of network areas  by noninvasive or invasive stimulation technologies may improve clinical symptoms.Conceptualization and study design: Shu Su, Runhua Sha, Jing Zhao, and Yingqian Chen; Methodology: Shu Su, Runhua Sha, Long Qian, Gerald L. Cheung, Jing Zhao, and Yingqian Chen; Investigation: Shu Su, Runhua Sha, Liping Lin, Haishan Qiu, Manshi Hu, and Chao Wu; Formal Analysis: Shu Su, Runhua Sha, Liping Lin, and Haishan Qiu; Writing—Original Draft: Shu Su, Runhua Sha, and Yingqian Chen; Writing – Review and Editing: Shu Su, Runhua Sha, Zhiyun Yang, Long Qian, Jianping Chu, Manshi Hu, Chao Wu, Yingqian Chen, and Jing Zhao; Visualization: Shu Su and Runhua Sha. All the coauthors listed above gave their final approval of this manuscript version.This study was supported by the Guangdong Basic and Applied Basic Research Foundation, China (No. 2021A1515012279) and Science, Technology Program of Guangzhou, China , the Natural Science Fund Youth Science Fund Project of China , and the Medical Scientific Research Foundation of Guangdong Province (No. A2020327). The authors of this manuscript declare no relationships with any companies whose products or services may be related to the article's subject matter.The authors of this manuscript declare no relationships with any companies whose products or services may be related to the article's subject matter.Written informed consent was obtained from all subjects.Appendix S1Click here for additional data file.


Refractory and relapsed acute myeloid leukemia (r/rAML) is associated with a difficult prognosis; clinical trials are typically suggested despite lack of a recognized standard of care. Combinatorial chemotherapy regimens utilized for r/rAML salvage play a crucial role in battling this invasive phase. Although it is characterized by a low response rate, CLAG is a traditional regimen used in r/rAML. We aimed to compare the efficacy and toxicity of CLAG+PLD to explore whether there was any improvement with the addition of pegylated liposomal doxorubicin (PLD) to CLAGA total of 110 r/rAML patients were retrospectively analyzed from February 2017 to June 2020 at the Medical Center of Hematology, XinQiao Hospital, the 303rd Hospital of the Chinese People's Liberation Army, and Central Hospital of Chang Sha, Hunan Province. The response, overall survival (OS), disease‐free survival (DFS), and side effects in 110 r/rAML patients were evaluated retrospectively. Of these, 55 patients were administered CLAG+PLD, while 55 patients received CLAG alone as salvage therapy.p = 0.023 and p = 0.045, respectively). The results of the Cox regression analysis for the CLAG+PLD group were strongly illustrative of the importance of hematopoietic stem cell transplantation (HSCT) following salvage therapy. No increased toxicity was observed in the CLAG+PLD group.In the CLAG+PLD group, there were 27 (49.1%) cases of complete response (CR) with no measurable residual disease (MRD−), 12 (21.8%) cases of CR with positive MRD (MRD+), 5 (9.1%) cases of partial response (PR), 11 (20%) cases of no response (NR), and no cases of death during the cycles. The response rates in the CLAG group were lower: CR was reached in 24 (46.6%) patients with MRD−, 6 (10.9%) patients with MRD+, 10 (18.2%) patients with PR, 13 (23.6%) patients with NR, and 2 (3.6%) patients who passed away, one from infection and the other from cerebral hemorrhage. The median OS and DFS were not attained in the CLAG+PLD group during the 2‐year OS and DFS follow‐up, while both values were 10 months in the CLAG group (CLAG+PLD is a potential salvage regimen for r/r AML that has a similar toxicity profile to CLAG and that improves response rates, 2‐year OS, and DFS relative to CLAG. R/rAML has a poor prognosis, and treatment is difficult. Primary refractory or resistant AML refers to the 10% to 40% of newly diagnosed AML cases that do not achieve complete remission (CR) following rigorous induction treatment.An analog named Cladribine is active against r/rAML. Pharmacological studies have demonstrated that cladribine before treatment promotes cytarabine intracellular absorption and cytarabine triphosphate (Ara‐CTP) buildup in AML blasts both in vitro and ex vivo.PLD, short for pegylated liposomal doxorubicin, exhibited a reduced cardiotoxicity and an improved pharmacokinetic profile compared to traditional doxorubicin applied in various malignancies.We have reported the application of CLAG+PLD in the successful treatment of a primary refractory blastic plasmacytoid dendritic cell tumors (BPDCN),22.1A total of 110 r/rAML patients were retrospectively analyzed from February 2017 to June 2020 at the Medical Center of Hematology, XinQiao Hospital, the 303rd Hospital of the Chinese People's Liberation Army, and Central Hospital of Chang Sha, Hunan Province. This was a retrospective matched case–control study involving two groups. We reviewed data and possible risk factors in these groups related to patient response in r/rAML. The controls in this matched case–control study were chosen based on factors that included the following: age, sex, leukocytes at diagnosis, ECOG, FAB classification, altered genes, risk stratification, and previous rounds of chemotherapy. CLAG+PLD was administered during the period of 2017 to June 2020. Patients in the control CLAG group were chosen retrospectively relative to the CLAG+PLD group according to the aforementioned matching rules. The follow‐up was 26 months (14.88–19.12), whereas, the follow‐up in the CLAG group was 16.2 months (13.39–19.01).The CLAG+PLD group and the CLAG group each consisted of 55 patients, all of whom met the following inclusion criteria: (1) a diagnosis of AML based on morphology, immunology, cytogenetics, and molecular biology; (2) relapsed or refractory AML with more than 10% leukemia blasts and (3) receipt of CLAG+PLD or CLAG treatment according to disease conditions, clinical need, and the patients' willingness. If multiple lines of salvage therapy were performed after enrollment, only the first line was recorded and analyzed, which meant no patients were included repeatedly. Risk stratification was evaluated according to the 2017 ELN AML Guidelines based on patients' cytogenetics and molecular abnormalities.The study protocol was approved by the Ethics Committee Review Board of the Second Hospital of Army Medical University. Since this was a retrospective analysis, a waiver/exemption regarding written informed was granted by the Ethics Committee.2.22 per week in multiple myeloma (MM).2 to minimize adverse effects. We propose to address the question of dose exploration in a subsequent prospective study.This is a retrospective study. The usual application dosages of PLD are 40 and 5 mg/m2 and cytarabine 1–2 g/m2 as a continuous infusion over the course of 5 days (cytarabine can be tailored according to the score of the Eastern Cooperative Oncology Group (ECOG)); G‐CSF priming 5 μg/kg (days 1–5), omitted if the leukocyte count was above 20 × 109/L; and PLD 30 mg/m2 as a continuous infusion. The CLAG group was treated with the classical CLAG regimen: cladribine 5 mg/m2/day (days 1–5); Ara‐C 2 g/m2/day (days 1–5); and G‐CSF priming 5 μg/kg (days 1–5), omitted if the leukocyte count was above 20 × 109/L.The patients in the CLAG+PLD group received the following regimen: cladribine 5 mg/mTarget drugs such as BCL‐2 inhibitor and FLT3 inhibitor were chosen according to the gene features at the re‐induction; however, the target drug was tailored, then quickly stopped when myeloid‐supression occurred. To be more specific, the average duration of treatment with the target drug ranged from 5 to 12 days.Consolidation after one cycle of CLAG+PLD or PLD was planned according to the patient's response and performance status. If HSCT was feasible and could be performed in a timely fashion, this was the preferred consolidation. If the patient was fit enough to receive a second cycle but HSCT was not planned or could not be scheduled until much later, a second cycle of chemotherapy was recommended.2.39/L; platelet count above 100 × 109/L; and lack of extramedullary disease. Partial remission (PR) was defined by a lack of extramedullary disease, along with any of the following: 5%–25% BM blasts, a decrease in BM blasts by at least 50%, or Auer rod positivity and <5% BM blasts. Aberrant leukemia‐associated immune phenotypes (LAIPs) detected by multiparameter flow cytometry (MFC) were primarily used to test for MRD in bone marrow samples; MRD‐positive status was defined as >0.01% of cells with a LAIP phenotype in bone marrow samples. The overall remission rate (ORR) was defined as the combined rate of CR and PR. Overall survival (OS) was calculated from the date of treatment to the time of death from any cause. DFS was defined as the time interval to the first event (relapse or death) and calculated from the date of treatment and the time of first event from any cause. The follow‐up duration was 24 months (range 0.5–45).CR was defined by the following criteria: bone marrow (BM) with at least 20% cellularity and under 5% BM blasts at steady state after chemotherapy, without cytological evidence of leukemia; no transfusion requirement; leukocyte count above 1 × 102.4The molecular abnormalities of all patients were analyzed before CLAG+PLD or CLAG salvage treatment. Genomic DNA was extracted using the EZNA Blood DNA Midi Kit (Omega Bio‐Tek). DNA samples were sequenced by next‐generation sequencing (NGS) using the MiSeq platform (Illumina); the target sequencing panel covered the entire coding sequences of 143 genes known to be relevant to AML pathogenesis, with an average effective depth ≥1000× in the target area.2.5χ2) test or Fisher's exact test was used for categorical variables, and the Kruskal–Wallis test was used for continuous variables. Survival functions were estimated using the Cox regression method and Kaplan–Meier (KM) method and were compared using the log‐rank test. All analyses were performed using GraphPad Prism 9, SPSS 21.0 software  and Office 2010 software . A two‐sided p value <0.05 was considered statistically significant.The data cutoff for this report was January 2020. Assessments at screening served as baseline data. Descriptive statistics are presented as the mean ± standard deviation (mean ± SD) for continuous data and as numbers and percentages for dichotomous/categorical data. The chi‐square , 6 (10.9%), 10 (18.2%), and 13 (23.6%) of patients, respectively (p = 0.007). Notably, 2 patients died in the CLAG group: one died of infection, and the other died of cerebral hemorrhage. Regarding overall survival (OS), the median survival was not reached in the CLAG+PLD group but was 10 months in the CLAG group, with a significance of p = 0.000. Similarly, the median disease‐free survival (DFS) was not reached in the CLAG+PLD group, while the value in the CLAG group was 10 months (p = 0.000)  Figure .3.3p = 0.010 and 0.017, respectively. Furthermore, the KM survival curves of the CLAG+PLD group showed significant differences in OS and DFS when stratified by the variables of response state and whether patients underwent HSCT after salvage reinduction with CLAG+PLD . Notably, 2 patients in the CLAG group died: one from infection and the other from cerebral hemorrhage. Other nonhematologic toxicities, including alopecia, infection, nausea/vomiting, sepsis, diarrhea, pneumonia, and asthenia, were infrequent and were not significantly different between the CLAG+PLD and CLAG groups. Details regarding hematologic and nonhematologic toxicity during induction treatment are provided in Table All patients experienced WHO grade 4 granulocytopenia, anemia, and thrombocytopenia. The CLAG+PLD and CLAG groups did not differ in terms of minimal neutrophil, platelet, or hemoglobin recovery. Severe (grades 3–4) infections occurred in 33% and 28% of the patients in the CLAG+PLD and CLAG groups, respectively p = 0.5. Notably4Described in Table In this research, treatment with CLAG+PLD produced a higher CR rate and longer OS and DFS than CLAG Table  and 2. Ip = 0.007), indicating a better depth of remission.Consistently positive MRD after induction treatment is associated with a poor prognosis in acute lymphoblastic leukemia (ALL)p = 0.10 and 0.17). It is obvious that a better response is related to OS of r/rAML. HSCT still plays a vital role in the survival of patients with r/rAML. Although there was no significant difference in response status before HSCT in the CLAG+PLD group, HSCT extended both OS and DFS   Figure . Therefo2, not 2 g/m2 as in classical CLAG. Another aspect is that PLD exhibits reduced cardiotoxicity and an improved pharmacokinetic profile with milder myeloid suppression and a lower rate of infections than doxorubicin. The CLAG+PLD regimen may be safer for patients with heart dysfunction due to its milder cardiotoxicity.The overall toxic side effects of CLAG+PLD were not significantly greater than those of CLAG Table . PatientThis was a retrospective clinical study, with all the inherent limitations of such a design. First, the number of included patients was limited. Second, since a wide variety of agents are currently available; improving the response rate by combining our CLAG+PLD regimen with novel agents may be a potential treatment strategy for r/rAML. Thus, additional work is in progress to dissect the underlying biology in preclinical models and multicenter clinical trials.Han Yao: Conceptualization ; data curation (lead); project administration ; writing – original draft (lead). Cheng Zhang: Conceptualization . Xu Tan: Data curation ; formal analysis ; investigation . Jieping Li: Data curation ; project administration . Xiaolin Yin: Methodology . Xiaojuan Deng: Methodology . Ting Chen: Data curation ; formal analysis . Jun Rao: Investigation ; methodology . Lei Gao: Project administration ; supervision . Peiyang Kong: Conceptualization (lead); project administration . Xi Zhang: Conceptualization ; funding acquisition ; investigation .


We present a very rare case of spontaneous hemothorax which was caused by carcinoma of unknown primary. To the best of our knowledge, there are no other such cases reported worldwide. The patient was 72‐year‐old male who was referred to our department for massive hemothorax. We undertook surgical treatment for haemostasis, and found multiple tumours which led to the diagnosis of carcinoma of unknown primary as the cause of haemorrhage. The tumours increased and grew rapidly after surgery and hemothorax progressed despite our treatment. The patient died from disseminated intravascular coagulation caused by continuous bleeding on postoperative day 19. We present a case of spontaneous hemothorax which was caused by carcinoma of unknown primary. Since it was very rare cause of hemothorax, we had difficulty in deciding the course of treatment. Hemothorax is mostly caused by trauma or iatrogenic, and spontaneous hemothorax is much less common.A 72‐year‐old male patient with bilateral pleural effusions was transferred to our hospital by ambulance from a local hospital. He had a history of hepatitis C , and benign adrenal tumour. He used no antiplatelet or anticoagulant drugs. On arrival, his blood pressure was 140/81 mmHg, heart rate was 98 bpm, respiratory rate was 23 breaths/min, and oxygen saturation < 90% on oxygen administration at 2 L/min with a nonrebreather mask. Blood test results revealed haemoglobin 8.5 g/dL. Platelet count, protrombin time Figure , internaIntraoperatively, we found various sizes of multiple lesions, which looked like hematoma, adhering to the visceral pleura of the lung Figure . The exuAfter the operation, the pleural effusion of the right thoracic cavity gradually increased Figure  and haemThe histopathological diagnosis of the lesion was confirmed on postoperative day 11. It was a cystic lesion adhering to the visceral pleura Figure . Microsc9/L: 2, Prolongation of prothrombin time 3.7 s: 1, Plasma fibrinogen 0.87 g/L:1, D‐dimer level 324.7 μg/mL:2, total score 6 = overt DIC)  Figure . We asseSpontaneous hemothorax is sometimes caused by neoplasia, and among neoplasia, angiosarcoma, schwannoma, thymoma, hepatocarcinoma, vascular tumours, soft tissue tumours, germ cell tumours, lung cancer, and mesothelioma have been reported as a cause.Surgical treatment for massive hemothorax is generally recommended in case of hemodynamic instability and evacuation of over 1500 mL through the chest tube and more than 200 mL/h blood loss through the chest tube over the first hour.Carcinoma of unknown primary is generally treated with regimens comprising platinum salts, and the prognosis have been reported to be very poor  except for 10%–15% favourable‐risk subsets.In conclusion, we have experienced a very rare bilateral hemothorax caused by carcinoma of unknown primary. Because no other such cases have been reported in the past, it is difficult to plan the treatment. Further experience should be reported.None declared.The authors declare that appropriate written informed consent was obtained for the publication of this manuscript and accompanying images.


Findings here imply lingering of virus, SARS‐CoV‐2, in the body for months. Thus, Nitazoxanide and/or other anti‐viral medications might be potential options to combat long COVID. This could transform treatment for long COVID patients globally.Long COVID or post‐acute sequelae of COVID‐19 (PASC) continues to affect many people even after a relatively mild acute illness. Underlying causes of PASC are poorly understood. There is no particular treatment or management program developed yet. Thus, the possibility of well‐known, safe anti‐viral medications use against PASC is proposed here. Patient‐X Symptoms and Duration: Red lightning bolt points to the Nitazoxanide treatment time, which was the breakthrough for her recovery.  However, varying from 10% to 87% of the patients, depending on the study, continue to be troubled by a variety of symptoms for months, including upper/lower respiratory, gastrointestinal, neurological problems and fatigue , chills, sweat, and muscle pains. Her symptoms worsened in ~2 weeks with congestion in sinuses and swollen tonsils, leading to breathing difficulties, fever up‐to 40–41°C, chills, pools of sweat, significant chest pain, lungs feeling like burning, anosmia, and diarrhea  and lung specialists in February and March 2021, respectively, due to worsening upper and lower respiratory symptoms. She was prescribed further medications  and then PASC, which can be considered sufficient for diagnosis.Other medications  receptors, which are highly expressed on lungs, gastrointestines, and endothelial cells.2+‐activated‐Cl− channels (TMEM16A –Transmembrane member 16A) were blocked by Nitazoxanide, thereby bronchodilating contracted airways, aiding recovery from respiratory illnesses.Furthermore, Nitazoxanide decreased pro‐inflammatory cytokines TNF‐α, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, and IL‐10 productions in peripheral blood mononuclear cells, resulting in reduced viral protein accumulation and suppressed IL‐6 overproduction in mice, one of the main cytokine storm mediators in respiratory viral infections.Various randomized, double‐blind, placebo‐controlled clinical trials have shown that Nitazoxanide is a safe medication for COVID‐19 patients.+ T‐cells, CD38 in CD4+ and CD8+ T‐cells, and CD38 and HLA‐DR on CD4+ T‐cells were also decreased among patients treated with Nitazoxanide compared to placebo.Another trial showed clinical improvement on the need of oxygen supplement in ~3 days in moderate cases besides SARS‐CoV‐2 RT‐PCR negativity by Day 7 subsequent to decrease in viral load.At a retrolective study, 552 COVID‐19 outpatients were administered mainly Nitazoxanide with Azithromycin  and corticosteroid Prednisone.Despite the Nitro group attached to C5 position of the Nitazoxanide Thiazole ring, which might cause concerns for toxicity, Nitazoxanide is well‐known for its safety and tolerance at approved doses, including for children.3.5Pro, a three domain Chymotrypsin‐like‐Cysteine protease, which processes critical viral protein precursors, thereby blocks viral replication, including for SARS‐CoV‐2 variants.Currently, Paxlovid, Sotrovimab, Remdesivir, and Molnupiravir are the main authorized treatment options for COVID‐19.Prodrug nucleoside analogues, Remdesivir and Molnupiravir, are phosphorylated to active forms in host cells, get incorporated into RNA by RNA‐dependent‐RNA‐polymerase (RdRp), causing viral replication obstruction.Additionally, Acyclovir and its analogues were found effective against coronaviruses SARS‐CoV‐1, HCoV‐NK63, MERS‐CoV in vitro.Recent studies illustrated significant correlations across COVID‐19, PASC and herpesviruses' reactivation, including Varicella‐Zoster (causing shingles), Epstein–Barr, HSV‐1 or Cytomegalovirus, attributed to COVID‐19‐induced lymphopenia.Although no viral resistance is detected for Molnupiravir yet,4Besides inflammatory factors, SARS‐COV‐2 remnants lingering in the body for months in various patients leading to PASC is becoming more evident.Denise Stewart: Conceptualization; data curation; methodology; validation; visualization; writing – original draft; writing – review and editing.None.None.Written informed consent for publication of their data was obtained from both patients. The prescriptions and clinical investigations were performed by their doctors upon them seeking help voluntarily due to their symptoms. This is a retrolective and descriptive review of these patients' clinical records, therefore the protocol was not submitted to an ethics committee.The patients' doctors had prescribed all their medications.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.Appendix S1Click here for additional data file.


Angiosarcoma of the breast is a rare malignancy. There are little data evaluating the survival and estimating the prognostic factors. The best surgical management and the role of systemic adjuvant therapy remain ill‐defined. This study aimed to investigate the clinicopathological features, survival, and prognostic factors of breast angiosarcoma.The data on patients diagnosed with breast angiosarcoma were extracted from the Surveillance, Epidemiology, and End Results database (1975–2016). Univariate and multivariate Cox regression analyses were used to estimate the influential prognostic factors. The overall survival (OS) and disease‐specific survival (DSS) of patients with breast angiosarcoma were evaluated.p < 0.05). Multivariate analysis suggested that lesions of the right breast, poor differentiation, and advanced stage were independent risk factors for OS or DSS . Older age was a risk factor in OS, but was protective in DSS. In primary breast angiosarcoma, age, laterality, grade, and stage were independent prognostic factors in OS and DSS . Mastectomy was also a risk factor in DSS (p = 0.034). The proportion of patients with grade III and regional disease was larger in the mastectomy group.This study included 656 patients diagnosed with breast angiosarcoma between 1975 and 2016. The 5‐year OS rate of all patients was 44.9% (95% CI 40.8–49.0). In both OS and DSS, Kaplan–Meier survival analyses revealed significant differences for both OS and DSS according to age, year at diagnosis, laterality, grade, and stage (all log‐rank Angiosarcoma of the breast had a poor prognosis. In our study, age, laterality, histologic grade, and stage were identified as significant prognostic factors. Why patients with angiosarcoma of the right breast had a worse prognosis remains equivocal. Mastectomy was adopted more often by surgeons in this cohort study for patients with advanced primary breast angiosarcoma. These tumors can occur in different parts of the body.Angiosarcoma of the breast is an extremely rare and heterogenous soft‐tissue tumor, which accounts for <1% of total breast malignancies.The relevant literature that is available is mainly case reports, meta‐analyses, and single‐center retrospective studies with a limited number of cases. Thus, in the present study, we retrospectively analyzed the clinical data of larger‐scale breast angiosarcoma patients obtained from the Surveillance, Epidemiology, and End Results (SEER) database to study the survival outcomes and prognostic factors. This study strategy reflected the rarity of the disease, but the varied survival outcomes between primary and secondary tumors.22.1The data were from the SEER database of the National Cancer Institute. SEER*Stat, version 8.3.9 is highly accurate with high positive predictive value,2.2https://seer.cancer.gov/data‐software/documentation/seerstat/nov2018), the grade system was used to classify the tumor into well‐differentiated (grade I), moderately differentiated (grade II), poorly differentiated (grade III), undifferentiated (grade IV), or unknown based on ICD‐O‐3. Also in the dictionary, the SEER historic stage A was used to categorize the cases as local, regional, distant, or unknown, which is derived from Collaborate Stage and Extent of Disease.All patients were diagnosed from 1975 to 2016. Patients' clinicopathological features and survival data were extracted by International Classification of Disease for Oncology (ICD‐O) site recode 3 (breast) and histologic subtype ICD‐O‐3 (angiosarcoma 9120/3). The variables in the case listing included patient identification, age at diagnosis, sex, race recode, year of diagnosis, laterality, grade, stage, surgery, chemotherapy recode, radiotherapy recode, survival months, vital status, and cause of death to site recode. Considering the importance of predicting survival,2.3χ2 test was used to compare the differences in patient characteristics between the left and right sites. The χ2 test was also used to explore the differences in the constituent ratio of different literalities and different surgical methods in patients with primary breast angiosarcoma. Overall survival (OS) was defined as the time from diagnosis of breast angiosarcoma to death due to any cause or the follow‐up cutoff. Disease‐specific survival (DSS) was calculated from the diagnosis of breast angiosarcoma to death of this disease. Kaplan–Meier survival curve and log‐rank tests were conducted to compare OS and DSS among different groups. In the part of primary breast angiosarcoma, Kaplan–Meier survival curve, and log‐rank tests were used to compare the survival of patients with different surgical methods. For both OS and DSS, univariate and multivariate Cox proportional hazard models were performed to assess the influences of age at diagnosis, race, year of diagnosis, laterality, grade, stage, surgery, chemotherapy, and radiotherapy on hazard ratios (HRs). The 95% confidence intervals (CIs) were computed for 5‐year OS, 5‐year DSS, and HRs. All variables in our study were assigned with Microsoft Excel 2016 MSO (16.0.13901.20148) 32bit. All statistical analyses were performed using IBM SPSS Statistics Version 26 software. All figures were drawn by GraphPad Prism 8.0.2 software. A p‐value ≤0.05 was considered statistically significant. All tests in our study were 2‐tailed.Descriptive statistics were used to examine the baseline characteristics of the 656 patients with breast angiosarcoma. For the laterality group, the 33.1In the search of the SEER database, 656 patients met the inclusion criteria. The demographic and clinical characteristics are summarized in Table 3.2p < 0.05). Interestingly, there was also a statistically significant difference in OS between the left and right breasts (log‐rank p = 0.041)  (Table p = 0.86) or radiotherapy group (log‐rank p = 0.86)  Figure . The Kap) Figure . Compare) Figure .p = 0.006) and ≥80 years of age . Black race was also a significant independent risk factor of OS in patients with breast angiosarcoma . Consistent with the Kaplan–Meier survival and univariate analyses, the multivariate analysis revealed significant differences between patients with different laterality. The HR for the right lesion group compared with right group was 1.34 . In addition, taking the well‐differentiated group and localized group as the classification reference, grade, and stage were independent risk factors affecting prognosis. HR was 2.17  for patients with grade III disease and 1.98  for those with grade IV disease. The prognosis was dismal for patients with regional disease , and distant disease had performed dismal prognosis .In multivariate analysis Table , regardi3.3p < 0.05). Race did not show statistical differences, which was inconsistent with the OS data (log‐rank p = 0.802). The 5‐year DSS of patients with higher age was more optimistic. Compared with patients ≤39 years , the 5‐year DSS was 79.9% (95% CI 70.9–88.9) in patients 60–69 years of age, 94.8% (95% CI 90.7–98.9) in patients 70–79 years, and 90.0% (95% CI 83.3–96.7) in patients ≥80 years (Table The 5‐year DSS of the entire cohort was 78.3% (95% CI 74.2–82.4) Figure . In the rs Table .p = 0.015). Older age seemed to be a protective factor in DSS. DSS benefits were evident in patients aged 60–69 years , 70–79 years , and ≥80 years . Although Kaplan–Meier survival analysis showed that patients who received aggressive treatment tended to associate with worse survival outcomes . Multivariate analysis (Table p = 0.034). Patients ≥80 years of age with right lesions and higher and more severe stages were associated with worse survival. Compared with the mastectomy group, the Kaplan–Meier survival analysis in OS and DSS  and DSS (log‐rank p < 0.001). Constituent ratio and chi‐squared test data (Table p = 0.006) and more severe stage . In addition, in primary angiosarcoma, laterality remained a factor affecting the prognosis, prompting further analysis of the constituent ratios. Consistent with the previous results, the difference between the constituent ratios of the two groups was not statistically significant .p = 0.032) and secondary breast angiosarcoma .We observed opposite influences of age for OS and DSS. Older age was a risk factor in OS, but was protective in DSS. A systematic review of 222 patients diagnosed with radiation‐associated breast angiosarcoma after breast cancer (secondary breast angiosarcoma) from 74 articles revealed that age was a significant prognostic factor on OS  and DSS . The same result was evident in the analysis of primary breast angiosarcoma. The χ2 test was performed to further compare the characteristics and differences between the left and right groups. No statistically significant difference was found. Due to the rarity of the disease and the lack of some data from SEER database , our comparisons of patient characteristics were limited. The influence of laterality on patients with breast angiosarcoma has hardly been studied. Therefore, we cannot state whether our discovery is universal or not based on the results of other studies. Nevertheless, we tried to evaluate the result compared to the effect of laterality on breast cancer. Compared with patients irradiated for right‐sided breast cancer, patients with left lesions have been reported to have worse survival because of higher cardiac radiation dose.p = 0.331).p = 0.0491).p = 0.0136) in patients with breast cancer localized in the left breast.p = 0.014).Surprisingly, we found that right lesion was a significant risk factor for patients with breast angiosarcoma in OS .p < 0.001) in median OS between localized grade I and grade II (both not reached) versus grade III (36 months).p = 0.015).p < 0.001).In some early studies, the predictive effect of grade on prognosis was not so clear. A retrospective analysis of 49 cases with primary breast angiosarcoma did not reveal statistically significant differences between grade and the rates of local recurrence or distant metastases (p = 0.034). This was not convincing data, because only 15 primary breast angiosarcoma patients did not receive any surgery. This reflected the common use of mastectomy in patients with breast angiosarcoma. However, consistent with our results, no benefit of mastectomy on survival compared with breast‐conserving surgery was found in three studies.There is no established optimal treatment program for patients with breast angiosarcoma. Especially for patients with primary breast angiosarcoma, clinicians usually deliver different degrees of radiotherapy or chemotherapy on the basis of surgical intervention with negative margins according to their own experience. In our study, surgery, chemotherapy, or radiotherapy were not significant prognostic factors in multivariate analysis. The trend in the Kaplan–Meier survival curve of DSS was even less optimistic. Patients who received more aggressive treatment (including both surgery and systemic adjuvant therapy) displayed a trend to worse DSS Figure . In our p = 0.043).The effects of systemic adjuvant treatment (including chemotherapy and radiotherapy) for patients with breast angiosarcoma are being studied. In the analysis of 58 patients with nonmetastatic breast angiosarcoma; chemotherapy led to a significantly better OS in secondary breast angiosarcoma group ; formal analysis ; investigation ; methodology ; writing – original draft (lead); writing – review and editing . Yunhai Li: Conceptualization ; formal analysis ; investigation ; methodology ; writing – original draft (supporting); writing – review and editing . Yuanyuan Wang: Software . Zhao Li: Software . Huan Zhang: Resources . Yidan Gao: Resources . Jinxiang Tan: Conceptualization ; writing – review and editing .This study was supported in part by the National Natural Science Foundation of China (No. 81102008) and the Chongqing Human Resources and Social Security Bureau returned overseas students selected funding project (2013012). The funding source had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.The authors declare that they have no competing interests.Not applicable.Table S1.Click here for additional data file.


Alstonia boonei is used folklorically for the management of the multitude of conditions including cataract, which accounts for 50% of cases of blindness in the region. The current study set out to probe the traditional use of the aqueous extract of Alstonia boonei stem bark (ABE) as an anticataract remedy using Sprague Dawley rat models. We investigated the probable phytochemical constituents in the extract, in vitro antioxidant potential, and its in vitro aldose reductase inhibition. For the anticataract investigations, diabetic cataract was induced using galactose in 3-week-old Sprague Dawley rats, and age-related cataract was induced by the administration of sodium selenite to 10-day-old rat pups. Cataract scores in both models were determined after treatment with 30, 100, and 300 mgkg−1 doses of ABE and 10 mlkg−1 of distilled water. Lens glutathione, total lens protein, soluble lens proteins  crystallin, and aquaporin 0 levels in the enucleated lens homogenates were determined. Changes in lens to body weight were also determined with histopathological analysis done on the lenses in the selenite-induced cataract model. The presence of alkaloids, tannins, flavonoids, glycosides, and triterpenoids was identified in the extract. The extract inhibited aldose reductase activity with IC50 of 92.30 μgml−1. The 30, 100, and 300 mgkg−1ABE-treated rats recorded significantly (p < 0.05) reduced cataract scores indicating a delay in cataractogenesis in galactose-induced cataract and in selenite-induced cataractogenesis as well. Markers of lens transparency such as AQP0, alpha-A crystallin, and total lens proteins and lens glutathione levels were significantly (p < 0.05) preserved. In conclusion, this study establishes the anticataract potential of the aqueous stem bark extract of Alstonia boonei in Sprague Dawley rat models.In Africa,  Cataracts are described as lens clouding that results in visual impairment , 2. In AAge-related cataract remains a leading cause of blindness worldwide and is believed to increase the risk of death . MultiplTo eliminate cataract, the WHO launched the Vision 2020 campaign , which cAlstonia boonei although there has not been any scientific justification of its use in that regard.One of the known alternatives to surgery largely employed in cataract treatment in Africa is the use of plant and plant-based products . One sucAlstonia boonei De Wild (Apocynaceae) is a large tree commonly found in the dry lowlands and rainforests across West Africa. The stem bark has a myriad of uses in folklore medicine, including its use as antimalarial and antivenom [Alstonia boonei in galactose- and selenite-induced cataracts in Sprague Dawley rats and provide scientific evidence to its traditional use.ntivenom  and antintivenom . The curAlstonia boonei stem bark was collected from Asakraka-Kwahu in the Eastern Region of Ghana in November 2020 and validated at the Department of Herbal Medicine, KNUST (Voucher specimen No: KNUST/HMI/2022/SB002). The plant material was then sun-dried.Sprague Dawley rats (3 weeks old and 10 days old) of both sexes were procured from the Center for Plant Medicine Research (CPMR), Mampong, Eastern Region, Ghana, and housed in stainless steel cages at the Department of Pharmacology Animal Housing Facility, UCC. Rats were allowed free access to commercial chow and water. Animals were humanely handled throughout the entire duration of the studies.Acetic anhydride, ammoniacal alcohol, ammonia (liquid), chloroform, Dragendorff's reagent, hydrochloric acid, Mayer's reagent, Fehling's solution A &B, ferric chloride, lead acetate, reduced nicotinamide adenine dinucleotide phosphate (NADPH), sodium selenite, sulphuric acid, and sodium hydroxide were purchased from Sigma-Aldrich, Germany; galactose was purchased from ACROS, New Jersey, USA; tropicamide ophthalmic solution (1%) was purchased from Alcon Laboratories South Africa (Pty) Ltd.Alstonia boonei was blended using a heavy-duty blender (37BL85 (240CB6) Waring Commercial, USA). The fine powder obtained was transferred into a percolator and macerated with 4.5 L of distilled water for 3 days. The mixture was filtered, and the filtrate was concentrated at 40°C using a rotary evaporator  and further concentrated at 50°C using an industrial oven . A brownish solid is produced which was stored at 4°C, reconstituted using PBS when needed, and referred to as Alstonia boonei extract (ABE) [About 640 g of the stem bark of ct (ABE) .The phytochemical contents of ABE were studied using methods as described by Harborne .2 and 0.3 ml of 10% AlCl3 was prepared. The mixture was incubated at 25°C for 30 min in a laboratory incubator , 2 ml of 1 moll−1 NaOH was added, and absorbance was determined at 415 nm using a spectrophotometer . A standard curve was established with quercetin, and the total flavonoid capacity of the extract was extrapolated from the curve.The method described by Chang et al.  was usedThe Folin–Ciocalteu method as described by Hudz et al.  was used3 was added afterwards. The resulting solution was further incubated at ambient temperature for 90 min, and the absorbance was determined at 760 nm using a spectrophotometer .A 0.6 ml mixture containing 0.1 ml of 0.5 N Folin–Ciocalteu and 0.5 ml of ABE was prepared and incubated at 25°C for an hour, and 2.5 ml of 2% NaHCOTotal antioxidant capacity estimated as gallic acid equivalent (GAE) was determined as earlier described by Urbano et al. . A finalμgml−1 in methanol) was added to a 20 mgl−1 mixture of methanol and DPPH. The resulting solution was incubated  under shade for 30 min at room temperature, and the absorbance was determined afterwards at 517 nm , using ascorbic acid  as the positive control. The IC50, the concentration at which there was a 50% decrease in the absorbance of DPPH, was then determined using the relation:The method described by Sharma and Bhat  was usedZ 36HK, Schaffhausen, Switzerland) at 5000 × g for 30 min at 4°C [μgml−1) were prepared as inhibitors of AR activity using phosphate-buffered saline (PBS). To 50 μl of the enzyme solution in a cuvette, 50 μl ABE, 50 μl 0.1 M phosphate buffer (pH 7), and 50 μl 0.03 mM NADPH were added. The enzyme reaction was initiated with the addition of 50 μl D-xylose to the mixtures, and absorbance was determined at 340 nm after 180 s using BioTek 800 TS absorbance reader . The inhibition of AR activity was expressed as a percentage of quercetin [In this assay, unrefined aldose reductase (AR) in enzyme solution was collected as the supernatant from a homogenized rat lens which was centrifuged (Bioswisstec Hermle n at 4°C . Varyinguercetin .n = 5) and treated as indicated below for 6 weeks: Group 1 received only 10 mlkg−1 distilled water p.o. for 6 weeks; Group 2 received 3000 mgkg−1 galactose p.o. daily plus 10 mlkg−1 distilled water p.o. for 6 weeks; and Groups 3–5 received 3000 mgkg−1 galactose p.o. daily plus 30, 100, or 300 mgkg−1 ABE p.o. for 6 weeks, respectively.The method used by Kyei et al.  and modiThe lenses of all rats used in the study were examined for degree of opaqueness weekly using a Macro II-B Slit Lamp  and scored based on the scale detailed  as descrBlood samples were taken, before the start of treatment and weekly, from the rat tail vein. All samples were taken after an overnight fast, and fasting blood sugar levels were determined using a glucometer .Rats were weighed before the study, and the lens was also weighed when extracted at the end of the study. The lens-to-body weight ratio was then computed.The extracted lenses were homogenized in PBS and centrifuged  at 5000 × g for 30 min to obtain a supernatant used for subsequent determinations. The supernatant obtained was used to determine GSH levels in the lens using a glutathione ELISA kit  in accordance with the guidelines provided by the manufacturer. The measurements were made in duplicate.Using a bicinchoninic acid (BCA) ELISA kit purchased from Shanghai Chemical Ltd, China, we determined the lens total protein from the supernatant using the manufacturer's guidelines. The measurements were made in duplicate.μmolkg−1 sodium selenite daily for two days and put into five groups (n = 4), and treatments meted out are detailed as follows: Group 1 received only 10 mlkg−1 distilled water p.o. and 12 hourly for 21 days without any sodium selenite being given to the rats; Group 2 received only 10 mlkg−1 distilled water p.o. 30 min before the sodium selenite challenge and 12 hourly for 21 days; and Groups 3–5 received 30, 100, and 300 mgkg−1 ABE p.o. 30 min before the sodium selenite challenge and 12 hourly for 21 days, respectively.We employed the model detailed by Kyei et al.  and modiThe pupils of the pups were dilated via ophthalmic tropicamide installation on day 22, and lenses were accessed for cataractogenesis using the slit lamp.The extent of cataract was graded as described by Sippel  and is dZ 36HK, Schaffhausen, Switzerland) at 5000 × g for 30 min, and the supernatant was collected for CRYAA and AQP0 assays using ELISA kits  in accordance with the manufacturer's guidelines. The measurements were all done in duplicate, and the experiment was repeated three times.Rat lenses were extracted at the end of the study and homogenized in PBS. The resultant solution was centrifuged . Values were considered statistically significant at ABE was realized to have detectable levels of alkaloids, flavonoids, glycosides, tannins, and triterpenoids .μgg−1 were realized for vitamin C, gallic acid, and quercetin, respectively.There was an observable increase in the total antioxidant capacity of all standards used with increased concentrations of ABE Figures . The ext50 of 92.30 μgml−1  drop in galactose levels at all dose levels studied over the 5-week period  rats were observed to show a steady level of blood galactose over the 5-week period. Upon galactose administration to rats, there was a steady rise in blood galactose level in the rats in the negative control (NeC) group. Treatment with ABE resulted in a significant  confirmed the observed blood galactose levels of 26.32 ± 0.55 in NC rats which increased to 72.59 ± 4.996 upon galactose administration in NeC rats. This rise in blood sugar was significantly reduced by ABE administration at doses of 30,100, and 300 mgkgectively .From the time-course curves obtained, galactose administration to rats caused an increase in cataract score which peaked after 1 week. However, ABE administration caused practically undetectable signs of cataractogenesis comparable with determinations made in NC rats .When the AUCs were determined, it established cataract score values spanning from 0 to 2 for ABE-treated rats as compared to about 13 in NeC rats .p < 0.05) increase in control rats exposed to galactose as compared to the distilled water-treated rats. Upon ABE treatment, rats recorded a significant (p ≤ 0.001) reduction in the lens-to-body weight ratio at 100 and 300 mgkg−1 doses administered  to 160.4 ± 13.71 ngl−1 when rats were challenged with galactose. Upon ABE administration, the GSH levels were significantly (p < 0.05) increased to 363.2 ± 58.73, 349.9 ± 42.61 ngl−1, and 392.1 ± 1.933 ngl−1 at doses of 30, 100, and 300 mgkg−1, respectively  decreased when rats were challenged with selenite to produce 15.43 ± 3.005 ngl−1 in NeC. When ABE  was administered, CRYAA levels were significantly (p < 0.05) increased to 59.76 ± 9.720 ngl−1, 69.66 ± 14.55 ngl−1, and 63.94 ± 7.361 ngl−1, respectively  decreased when rats were challenged with selenite. When ABE  was administered, AQP0 levels were significantly (p < 0.05) increased to 132.4 ± 2.163 ngml−1, 117.3 ± 8.140 ngml−1, and 144.7 ± 12.62 ngml−1, respectively  . In NeC Current understanding of the precise mechanism involved in the progression of diabetic cataract is still limited, leaving significant gaps in our knowledge. As a result, the development of reliable preventive and therapeutic medications for this condition is yet to be achieved. Nevertheless, the galactose cataract model replicates the occurrence of secondary cataract. Compared to glucose-induced cataract, galactose-induced cataract is efficient. The model is economical and can rapidly replicate the pathophysiology of diabetic cataract. Oftentimes, galactosemic cataract is used to investigate the mechanisms of action of drugs employed in the management of diabetes-associated complications. Moreover, galactosemic cataract develops quickly and is reversible compared to other models of diabetic cataract, such as streptozotocin and alloxan .α, β-γ crystallin [Galactosemic cataract mimics diabetic cataracts and is characterised by the detection of trace amounts of sugar in peripheral blood following the administration of galactose. These traces of sugar are instrumental to cataract development. On the other hand, selenite shots in the selenite cataract model, a replicate of age-related cataract, cause cataractogenesis by increasing oxidative stress due to increased levels of reactive oxygen species  and reduystallin  and distystallin .In the galactose cataract model, increased galactose consumption results in hyperglycemia which in a cascade reaction leads to upregulation of aldose reductase , 32, theIn contrast to the low affinity for glucose, aldose reductase has a higher affinity for galactose. Additionally, galactitol, the alcohol metabolite of galactose, has been shown to be much more challenging for sorbitol dehydrogenase to metabolize compared to sorbitol. Thus, galactosemia is more likely to cause severe cataracts in shorter time periods , 37. WitAlstonia boonei extract decreased cataract scores in rats. This observed effect can be attributed to the extract's inhibitory effect on aldose reductase, as well as the ability of the extract to prevent potential oxidative stress caused by the build-up of ROS when galactitol accumulates in the lens. In addition, this assertion is supported by the absence of physical signs of cataract, including an increase in lens weight and loss of body weight [Moreover, y weight . HoweverAmong the various antioxidants present in the lens, the tripeptide glutathione is the most abundant and plays a crucial role in the preservation of lens transparency . FurtherAlstonia boonei extract's inhibition of galactose-induced cataract, treatment with the extract also suppressed selenite-induced cataract in rat pups. This model serves as a valuable model for studying senile cataracts. It offers several advantages, including the ability to rapidly and consistently induce cataract formation within a short timeframe [α, β-γ crystallin [In addition to the imeframe . Due to imeframe . Signs oystallin .The preventive and therapeutic effects of phytoconstituents in age-related ocular disorders, particularly cataracts, have been extensively documented . FlavonoAdditionally, the extract demonstrated a preventive or delaying effect on selenite-induced cataracts in rodent pups. This outcome holds clinical significance as delaying the onset of cataract formation can help prevent the associated visual impairment that can impact the independence of affected individuals , 47. In Alstonia boonei extract could explain its preventive effect on senile cataract development.The impact of oxidative stress on selenite-induced cataracts is evident from the rapid emergence of nuclear cataracts within a maximum of 5 days following sodium selenite administration. Secondary metabolites from plants such as flavonoids and tannins possess antioxidant properties that enable them to eliminate ROS . The preSenile cataracts involve a cascade of biochemical processes, including a general reduction in protein levels and the insolubilization of soluble proteins within the eye lens . ProteinAlstonia boonei delays and prevents galactose and selenite-induced cataractogenesis in Sprague Dawley rats and pups, respectively. The observed anticataract activity may be attributed to the extract's ability to decrease oxidative stress in the lens. The current study gives scientific evidence to its claim as an anticataract agent. Future studies may, however, identify the specific phytochemical(s) accounting for the probable anticataract effect of the aqueous stem bark extract of Alstonia boonei.The aqueous stem bark extract of


Among cancers, gastric cancer has the fifth highest incidence worldwide and is the third most common mortality factor, which may have been due to inadequate knowledge of its molecular pathogenesis. The retinoblastoma gene (RB1), a tumor suppressor gene, may have a role in gastric cancer. This research aims to assess Rb expression as a prognostic marker to obtain more insight regarding gastric cancer.p < 0.05 was statistically considered meaningful.This retrospective analytical study was done on 61 patients  with gastric adenocarcinoma admitted from 2010 to 2012 in Shahid Sadoughi and Mortaz hospitals, Yazd, Iran. Demographic data, including age, gender, clinical signs and symptoms, and pathology reports, were retrieved from patients' hospital folders. Then, the altered Retinoblastoma gene expression was evaluated by immunohistochemistry studies. Acquired data were analyzed by SPSS software v.16. p > 0.05). There was only a meaningful relationship between the grade of tumors and survival rate (p = 0.039).In this study, the ratio of men to women was higher (2.81:1), and the mean age of patients was 62.44 years. About 90.2% of patients died during the study. There was no meaningful relationship between the presence of pRb, the intensity of staining, the percentage of staining with patients' age, gender, tumor grading, and survival rate (Altered pRB expression is not common in gastric cancer and does not impact the survival and grading of tumors. Poorly differentiated tumors had an ominous outcome with the lowest survival time. One of the cardinal cell cycle regulators in gastric cancer is the Rb gene pathway.Some data indicate that Rb expression induces a better prognosis in gastric cancer, while some findings suggest that it has no prognostic significance2This research was a retrospective and cross‐sectional descriptive‐analytical study of all patients with gastric adenocarcinoma admitted during 2010−2012 to Shahid Sadoughi and Mortaz hospitals in Yazd, Iran. Patients' missing details  and deaths due to causes other than gastric cancer were omitted from the criterion.Demographic information from the hospital ward archive, including age, gender, paraclinical information, surgery report, and pathology report, was retrieved. Then, appropriate gastric tissue blocks were chosen by evaluating hematoxylin and eosin stained slides in the pathology ward archive. Selected gastric tissue blocks preserved in paraffin were cut again, stained by immunohistochemistry (IHC), and then evaluated for the Rb gene.2.1Cancerous tissue samples were picked and sliced into 4‐μm‐thick pieces from paraffin‐embedded blocks. In xylene, they were de‐paraffinized, dehydrated by graded alcohols, and placed in 0.1% hydrogen peroxide to quench endogenous peroxidase activity. In a 10 mmol sodium citrate buffer , antigen retrieval was carried out using a 750 W microwave oven for 15 min. To avoid the nonspecific binding of antibodies, the sections were blocked with 10% normal goat serum for 30 min at room temperature. Inside a humidified chamber at 4°C overnight, the slides were incubated with anti‐human Rb monoclonal antibody . The sections were then incubated at 37°C for 30 min with a biotin‐labeled goat secondary anti‐rabbit antibody (Master polymer plus HRP), followed by the streptavidin‐biotin horseradish peroxidase complex reaction. The reaction products were detected by immersing the slides in a freshly formulated diaminobenzidine solution for 10 min before dehydration and mounting and by counterstaining them with hematoxylin.All slides were assessed under low magnification (×4) and then confirmed under high magnification ×20 and ×40) by light microscopy scanning the whole tissue specimen. A professional pathologist, blinded to the study design, assessed the staining severity. The immunostaining for RB protein was considered positive if there was brown nuclear staining of the neoplastic cells. So, normal and aberrant pRB expressions were determined for invasive carcinoma cases. Normal pRB expression was reflected by nuclear staining in all areas of tumor tissue with cell‐to‐cell heterogeneity and variable staining intensity. pRB expression was considered aberrant if the whole section or major focal areas within the section showed no nuclear staining with preserved reactivity in adjacent non‐neoplastic tissue. Samples were graded in three ways: positive/negative for the mutated Rb gene and stained tissue intensity  Figures  and 2. T0 and ×402.2T‐test, χ2 test, and ANOVA test. P Values lower than 0.05 were considered significant.All data was put into the program SPSS v.16  and analyzed by 3Of the 61 census‐studied patients, 45 (73.7%) were male, and 16 (26.3%) were female . The youngest patient was 21 years old, and the oldest was 91. For males, the average age was 64.13 year old, 57.68 year old for women, and 62.44 year old for all patients. Most patients  were 50 to 80 years of age. Fifty‐five patients (90.2%) had died within 5 years of diagnosis due to gastric cancer, and six patients (9.8%) were still alive.p = 0.272). There was also no relationship between age and the pRb expression (p = 0.57). Following block staining, 43 patients (70.5%) did not show positive stains, and 18 patients (29.5%) had positivity for pRb IHC‐staining. Eight (17.8%) of male patients had poorly stained tissue severity, and seven (15.6%) had strong intensity. No relationship between gender and the staining intensity of the tissue was found based on these results (p = 0.513). The average age of patients who do not have a mutant pRb is 61.67. As shown in Table p = 0.988). In poorly differentiated tumors, 18 (60%) tumors had no altered pRb expression, and 12 (40%) tumors had a pRb mutation. This data found no relationship between grading and the pRb mutation (p = 0.064). Thirty‐nine patients (90.7%) with pRb mutation died from gastric cancer. There was no relationship between the pRb mutation and the 5‐year survival of patients (p = 0.829). Two patients with well‐differentiated tumors (18.2%), four patients with moderately differentiated tumors (20%), and no one with poorly differentiated tumors was alive after 5 years (Details are shown in Table p = 0.039), that the less differentiation, the worse survival was seen.As shown in Table p = 0.359). Forty out of 45 male patients (89.9%) and 15 out of 16 female patients (93.7%) died from cancer because there was no gender‐to‐survival relationship (p = 0.575). Also, there was no correlation between patient age and survival (p = 0.700) .Further investigations are required to determine the Rb gene correlation to the grade and stage of tumors.Data regarding the Rb gene and its prognosis relationship varies, some indicating that Rb has a prognostic value, while some deny this.p = 0.829) and would not contribute to 5‐year survival. Tumor grading is the only prognostic factor in the current analysis; thus, survival drops with lower differentiations (p = 0.039).Feakins et al. and Song et al. also claim a poorer prognosis for tumors that produce less pRb.The limitation of our study was the small sample size and the loss of some samples due to lack of access to patients or lack of some considered variables in patients' files.5The presented data indicates that gastric cancer is more prevalent in males than females, but age, gender, altered pRb expression, and survival are not correlated. In 29.5% of tumors, the altered pRb expression is shown and is thus not a prevalent phenomenon in gastric cancer. Furthermore, there is no association between altered pRb expression and patient prognosis. The degree of altered pRb expression and the proportion of mutated cells do not significantly impact prognosis. However, tumor grading is linked to patient survival, with lower differentiation resulting in reduced 5‐year survival rates. Altered pRb expression, staining intensity, and the percentage of mutated cells do not correlate with tumor grading. More extensive sampling is necessary to identify the prognostic value of altered pRb in gastric adenocarcinoma neoplastic cells and other factors that may contribute to disease progression. Additionally, a genomic‐level examination is advised. Further research on gene mutation's influence on angiogenesis and vascular invasion and its potential impact on treatment sensitivity or resistance would be beneficial.Shokouh Taghipour Zahir: Conceptualization; formal analysis; supervision; validation; writing—original draft; writing—review and editing. Seyyed Hossein Razavi: Conceptualization; investigation; project administration; writing—original draft; writing—review and editing. Farzan SafiDahaj: Formal analysis; methodology; project administration; writing—original draft; writing—review and editing. Koorosh Rahmani: Investigation; project administration; writing—original draft; writing—review and editing. Sajad Sadeghinejad‐Alamabadi: Investigation; methodology; project administration; writing—original draft; writing—review and editing.The authors declare no conflict of interest.This research was analyzed and obtained ethnic code by the Yazd Shahid Sadoughi Medical Science University ethnic commission (IR.SSU.MEDICINE.REC.1396.37). The authorization to view patient records and gather the required data was collected from medical centers. A trained medical student telephoned the family of patients to obtain survival information. During and after the research, the patient's details were confidential.The lead author Sajad Sadeghinejad‐Alamabadi affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.


It is unclear whether long‐term heavy alcohol use leads to early cardiac function decline.Long‐term heavy alcohol use developed reduced cardiac function in subclinical status by analyzing myocardial work (MW). Epicardial adipose tissue (EAT) volume and serum biomarkers contribute to identify potential factors sensitive in predicting early cardiac function decline.We enrolled 31 asymptomatic participants with heavy alcohol use and 33 age and sex‐matching nondrinking individuals. Participants underwent echocardiography, MW analysis, EAT volume measurement, serum biochemical examinations, and body composition assessment. We used multivariate linear regression to identify correlation between MW and total cholesterol (TC), EAT volume, and placental growth factor (PlGF). To determine global work efficiency (GWE) below the normal reference value of 96%, we developed receiver operating curves with area under curve (AUC) to compare different combinations of TC, EAT volume, and PlGF.p = .004). TC was positively associated with GWE , whereas EAT volume  and PlGF  were inversely associated with GWE. The most significant AUC for reduced GWE was TC + EAT volume .All 64 participants were male. GWE was reduced in the alcohol use group compared with the control group (96, interquartile range [IQR] = [95–97.75] vs. 97, IQR = [97–98], Asymptomatic heavy alcohol use has shown early reduced cardiac function which can be associated with altered fat metabolism, suggesting individuals with alcohol use and abnormal fat metabolism need to be alert to heart damage. This cross‐sectional study showed that participants with heavy alcohol use but no cardiovascular disease symtoms experienced lower GWE than the control group, and the GWE correlated with TC, EAT volume, and PlGF. TC combined with EAT volume had a satisfactory prediction effect for GWE under 96% among all participants. Moreover, we applied various clinical examinations to find parameters correlated with early cardiac function change as detected by MW analysis, including epicardial adipose tissue (EAT) volume measurement, serum biochemical examinations, and body composition assessment. Total cholesterol (TC), EAT volume, and placental growth factor (PlGF) in the alcohol use group were examined in the study as potential independent variables to predict the global work efficiency (GWE) below the normal reference value of 96%.The first aim of this study was to figure out whether asymptomatic heavy drinkers developed reduced cardiac function in subclinical status by analyzing MW. The second aim was to identify potential factors sensitive in predicting early cardiac function decline in individuals with heavy alcohol use.22.1Participants in this study were recruited from residents who live near a distillery in Hebei Province in 2020. Recruitment information was shared through telephone communication. Inclusion criteria required participants aged over 18 with heavy alcohol use for more than 10 years, but with no cardiovascular disease symptoms. Heavy alcohol use was defined as over 14 drinks per week.2.2We collected personal alcohol use habits of participants in the alcohol use group using a questionnaire, which inquired about types of alcoholic beverages, weekly alcohol use frequency, and the amount of alcohol consumed each time. To calculate the amount of alcohol consumption (g per week), 100 mL of liquor, beer, and wine were assumed to contain 31.5, 9.46, and 3.94 g of pure alcohol, respectively, based on the average alcohol concentration of each type available in the market. Weekly pure alcohol intake was calculated as frequency (per week) × amounts (L) × pure alcohol (g/L). The result was presented as standard drinks per week.2.32 or relative wall thickness (RWT) over 0.51. The Chinese male thresholds of LVMI and RWT were defined by the Echocardiographic Measurements in Normal Chinese Adults (EMINCA) study.Echocardiographic examination was performed using Vivid E9 ultrasound system (GE Vingmed Ultrasound AS) with an M5Sc probe by an experienced practitioner. Left ventricular (LV) systolic function was assessed by LV ejection fraction (LVEF) using Biplane Simpson method and global longitudinal strain (GLS). GLS was reported as an absolute value. Cardiac remodeling was primarily assessed by LV mass index (LVMI) over 109 g/m2.4Global and each LV segment MW were measured by vendor‐specific software . An experienced sonographer who was blinded to the group of participants performed the analysis. The calculation of MW required LV longitudinal strain, peak systolic LV pressure, and setting valvular event times. Brachial artery systolic blood pressure by cuff method was assumed to be equal to peak systolic LV pressure. LV longitudinal strain was measured by speckle tracking echocardiography which needed 3 cardiac cycles each for 3, 4, and 2 chamber apical views with 60–80/s frame rates. Valvular event times including mitral valve closure, aortic valve opening, aortic valve closure, and mitral valve opening were set using the apical three‐chamber view. The software provided a personalized LV pressure‐strain loop for each subject. The segmental MW was shown in a bull's eye plot. Two global MW indices were calculated as an average of segmental values: Global constructive work (GCW) was the average work during shortening in systole, indicating the MW contributed to pumping blood; Global wasted work (GWW) was the average work during lengthening in systole and shortening in isovolumic relaxation, indicating the MW not contributed to pumping blood. Global work index (GWI) was the area of the LV pressure‐strain loop. GWE was calculated as GCW divided by the sum of GCW and GWW. If every LV segment shortens during systole, the GWE will be 100%.2.5Cardiac magnetic resonance  was used to measure EAT volume. An experienced radiologist traced out the contour of EAT compartments between visceral pericardium and myocardium using software (MATLAB 2014) and calculated the volume of EAT. EAT volume index was calculated as EAT volume divided by body surface area (BSA).2.62) = 186 × [serum creatinine (μmol/L)/88.4] −1.154 × age (years) −0.203.All participants received serum biochemical examinations. Parameters in this study included: TC, low density lipoprotein cholesterol (LDL‐C), triglyceride (TG), uric acid (UA), albumin, pre‐albumin, thyroid stimulating hormone (TSH), adiponectin, lipocalin2/NGAL, resistin, plasminogen activator inhibitor‐1 (PAI1), and leptin. HOMA‐IR and HOMA‐β were used to assess insulin resistance according to the formula: HOMA‐IR = fasting insulin × fasting blood glucose/22.5, HOMA‐β = 20 × fasting insulin/(fasting blood glucose‐3.5). PlGF and vascular endothelial growth factor A (VEGFA) were detected for angiogenesis. Chronic inflammation was evaluated by the level of high‐sensitivity C‐reactive protein (hsCRP) and interleukin‐6 (IL‐6). Renal function was calculated according to the four‐variable modification of diet in renal disease equation: glomerular filtration rate . The body composition parameters included in the study were body mass index (BMI), visceral fat area , visceral fat mass , and subcutaneous fat mass .2.8t‐test or the Mann–Whitney U‐test, as appropriate. Categorical variables were compared using Chi‐square test or Fisher exact test.The Shapiro–Wilk was used to test the normality distribution of raw and log‐transformed continuous variables. The central tendency and dispersion of normally distributed variables were described using the mean ± standard deviation, while nonnormally distributed variables were described using the median and interquartile range (IQR). Categorical variables were expressed as frequency and percentage. To test the existence of significant differences between the alcohol use group and the control group, continuous variables used unpaired Student p < .1) and clinical interest to make the regression result reliable.The correlation between MW and EAT volume and serum biomarkers in asymptomatic heavy alcohol use was tested using Spearman correlation coefficient and multivariate linear regression. Since the sample size of the alcohol use group was 31, we decided to select only 1–3 independent variables based on significant Spearman coefficient  with area under curve (AUC) were developed to compare different combinations of selected independent variables including TC, EAT volume and PlGF. Statistical analyses were performed using IBM SPSS Statistics for Windows, Version 22 software (IBM Corporation). All tests were two‐sided and their level of significance was set as 33.1p < .05).This study enrolled 31 participants with asymptomatic heavy alcohol use as the alcohol use group and 33 age and sex‐matched participants without alcohol use as the control group. The demographic characteristics of all participants are shown in Table 3.2The conventional measurement showed a significant difference in IVSd, LVPWd, LVM, LVMI, and RWT, indicating a tendency of cardiac remodeling in the alcohol use group.p = .004). No significant differences were found in GWI, GCW, and GWW between the two groups (p > .05)  Table .3.3n = 31). The positive results of the correlational analysis were presented in Table r = −.364, p = .048) and LDL‐C  showed negative correlations with GCW. HOMA‐β is negatively correlated with GWI  and GCW . No significant correlation was found in other clinical characteristics with global MW indices. Table Correlation and regression analysis was conducted to identify the biomarkers covariation with global MW in the alcohol use group n = 1. The poβ = .434, 95% confidence interval [CI] = 0.228 to 1.328, p = .008), whereas EAT volume  and PlGF  were inversely associated with GWE.Because reduced GWE in the alcohol use group had statistical significance, we chose GWE as the dependent variable in the regression analysis. TC, EAT volume, and PlGF were selected as independent variables for the model. As shown in Table 3.4n = 64). AUC for all combinations was presented in Table p = .006). Another interesting result was that the AUC of TC + BMI  showed no significant difference with TC + EAT volume (p = .6528). Table We combined TC with covariates mentioned above and body composition parameters including BMI, VFM, SFM, and VFA to test the prediction effect of different combinations for reduced GWE. Figure 4This cross‐sectional study showed that participants with heavy alcohol use but no cardiovascular disease symtoms experienced lower GWE than age and sex‐matched control group, and the GWE correlated with TC, EAT volume, and PlGF. TC combined with EAT volume had a satisfactory prediction effect for GWE under 96% among all participants while TC combined with BMI also showed a statistically similar result.Our study confirms that fat metabolism may be associated with altered cardiac function in heavy alcohol use. Recently, fat is regarded as an active endocrine organ that could profoundly influence metabolism.EAT is fat depots adjoining the myocardium without a barrier, allowing for more convenient cell communication between EAT and myocardium. EAT covers 80% of the heart surface, mainly in right ventricle surface, anterior wall of the left ventricle, and anterior and lateral walls of right atrium. EAT is composed of adipocytes, lymphocytes, and macrophages.Interestingly, PlGF is related to GWE in this study. PlGF is a member of VEGF family synthesized from placental, playing an important role in vascular inflammation and adverse outcome in patients with acute coronary syndrome,MW analysis showed an earlier cardiac function change on GWE and an increasing tendency of wasted work in the alcohol use group. At the same time, the conventional echocardiography showed no significant EF change but the heart was in the progress of cardiac remodeling. This result indicated that MW analysis was a sensitive method to help individuals with heavy alcohol use but no cardiovascular disease symptoms to detect reduced cardiac function. For better screening of reduced cardiac function among the population, we did ROC curves of common clinical examination parameters to determine GWE under 96%. TC + EAT volume had the most significant AUC and TC + BMI also showed good prediction value. By comparing the AUC of TC + EAT volume and TC + BMI, we found that the two combinations have statistically the same prediction efficacy. Among the population, lower TC and elevated BMI imply reduced cardiac work efficiency, especially in long‐term heavy alcohol use. Prior studies have noted the importance of alcohol abstinence to prevent ACM advance.This study has several limitations: First, this research had a limited sample size for both the alcohol use and the control groups. Only 31 participants met the inclusion criteria of the former from 250 volunteers. Second, the participants were all Chinese from northern China. There might be geographical bias and ethnic bias.5The purpose of the current study was to determine reduced cardiac function in individuals with heavy alcohol use but no cardiovascular disease symptoms using MW analysis, and to find out which parameters commonly used in clinical examinations are sensitive in predicting early cardiac function decline. The study has shown that GWE is significantly reduced in heavy alcohol use and correlates with TC, EAT volume, and PlGF. Further ROC curves tested the prediction ability for reduced GWE by combining TC with different variables. TC + EAT volume and TC + BMI have significant AUC.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.