Glucose can be involved in metabolic activities as a structural substance or signaling molecule and plays an important regulatory role in fruit development. Glucose metabolism is closely related to the phenylpropanoid pathway, but the specific role of glucose in regulating lignin biosynthesis in pear fruit is still unclear. The transcriptome of pear calli generated from fruit and treated with glucose was analyzed to investigate the role of glucose in lignin biosynthesis.PbPAL, PbHCT, PbCOMT, PbPRX) in lignin biosynthesis was up-regulated after glucose treatment. In addition, glucose might regulate lignin biosynthesis through interactions with ABA, GA, and SA signaling. Several candidate MYB transcription factors involved in glucose-induced lignin biosynthesis have also been revealed. The qRT-PCR analyses showed that the expression pattern of PbPFP at early developmental stage in ‘Dangshansuli’ fruits was consistent with the trend of lignin content. Transient expression of PbPFP resulted in a significant increase of lignin content in ‘Dangshansuli’ fruits at 35 days after full bloom (DAB) and tobacco leaves, indicating that PbPFP (Pbr015118.1) might be associated with the enhancement of lignin biosynthesis in response to glucose treatment.The treatment of exogenous glucose significantly enhanced the accumulation of lignin in pear calli. A total of 6566 differentially expressed genes were obtained by transcriptome sequencing. Glycolysis was found to be the pathway with significant changes. Many differentially expressed genes were enriched in secondary metabolic pathways, especially the phenylpropanoid pathway. Expression of structural genes (PbPFP plays a positive role in regulating lignin biosynthesis in response to glucose treatment. This study may reveal the regulatory pathway related to lignin accumulation in pear calli induced by glucose.The online version contains supplementary material available at 10.1186/s12870-022-03658-x. Lignin is a complex aromatic polymer and one of the important components of the secondary cell walls in plants . In vascAs previous studies, sugar metabolism exists in the whole process of fruit development and is an important factor affecting the intrinsic quality of fruit –10. Ther14 was introduced into a spruce twig by infusion, lignin in the cambium was radioactive, indicating the ultimate organic source of lignin was D-glucose [Some research showed that carbon source of lignin biosynthesis was provided by glucose . After D-glucose . The con-glucose . It prov-glucose . In summArabidopsis seedlings stopped growing, while the effect can be reversed with exogenous ethylene [In addition, the regulatory effects of glucose are usually accompanied by interactions with plant hormone signal transduction pathways. Accumulation of abscisic acid (ABA) and the expression of related genes could be significantly increased by glucose . For exaethylene , 30. SimArabidopsis seedlings [Moreover, the deposition of lignin occurs after the peak of sugar accumulation, and the amount of sugar accumulation directly affects the biosynthesis of lignin in pear fruits . Exogenoeedlings . Sugar aeedlings . HoweverIn order to identify the effect of glucose on lignin biosynthesis in pear flesh calli, the calli were transferred to MS solid medium with different concentrations of glucose , and the samples were collected at 15 d post-treatment. Lignin content of calli was significantly increased after treating with 1–4% glucose  and the control (CK) for transcriptome sequencing, then evaluated the quality of six cDNA libraries. The number of clean reads in each library ranged from 44 to 58 million, with an average Q20  of > 96% | ≥ 1.5. A total of 6566 DEGs were identified with 3829 up-regulated and 2737 down-regulated genes | ≥ 4, there were 1843 DEGs with 1025 up-regulated and 818 down-regulated genes. Moreover, when |log2(GLU vs CK)| ≥ 8, there were 723 DEGs with 413 up-regulated and 310 down-regulated genes  analysis was used to investigate the main functions of these specifically regulated genes. There were many DEGs related to biological processes and cellular components under glucose treatment. In the biological process category, most DEGs were associated with ‘cellular process’, ‘organic substance metabolic process’ and ‘primary metabolic process’. In the cellular component ontology, most DEGs were related to ‘intracellular’, ‘organelle’ and ‘intracellular organelle’. The GO term related to molecular function was significantly enriched in ‘structural molecule activity’ and ‘structural constituent of ribosome’ Fig. A. The reThen, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to analyze various metabolic pathways of DEGs to provide information and further understanding of how glucose regulates metabolism in cells and the synthesis of secondary metabolites, such as lignin, at the molecular level. We selected the top 25 KEGG enrichment pathways for analysis, including the carbon metabolism (250 DEGs), the plant hormone signal transduction (229 DEGs), glycolysis/gluconeogenesis (126 DEGs), mRNA surveillance pathway (122 DEGs) and phenylpropanoid biosynthesis (86 DEGs). Moreover, the expressions of genes involved in phenylalanine, tyrosine and tryptophan biosynthesis also changed Fig. B.PbHXK, and three DEGs were annotated as PbPK , whose expressions were up-regulated by glucose. Nevertheless, many DEGs were annotated into the PFK gene family, which had the largest number of DEGs in glycolysis, including PbPFK (6) and PbPFP (1)  and 1 PbPFP (Pbr015118.1) were all up-regulated in glucose-treated calli, suggesting that they might be the candidate genes for lignin biosynthesis enhancement in response to glucose.Sugar provides energy for metabolism and signaling molecules for plants . The genng PbPFK  and PbPFPbPAL (Pbr008363.1 and Pbr016460.1), PbCOMT  and some of Pb4CL (Pbr013445.1), PbHCT , PbC3H , PbPRX  were positively correlated with lignin biosynthesis in pear calli, and their expressions were significantly up-regulated under glucose treatment. However, the expressions of PbC4H , PbCCR , PbF5H  were down-regulated under glucose treatment. Nevertheless, these results indicated the activation of the phenylpropanoid pathway.The primary metabolism plays an important role in regulating secondary metabolism , and fluPbMYB (48), PbNAC (47), and PbWRKY (35) were the most enriched TFs in this study  , PbTIR1 (1), PbAUX/IAA (9), PbARF (5) and PbSAUR (6) genes were up-regulated after glucose treatment. ABA signaling is associated with plant secondary cell wall formation and lignin deposition [2 (GLU vs CK) fold changes of PbABF genes were more than 6, PbPP2C and PbSnRK2 genes were on the contrary. GA regulates lignin biosynthesis [PbDELLA (Pbr024549.1), other genes were down-regulated. Moreover, six DEGs of the PbPR-1 family were all up-regulated after glucose treatment in the SA signaling pathway.Plant hormones are essential components of the sugar sensing and signaling network that control plant growth and development , 42. DEGUR) Fig. . Among tposition . In ABA ynthesis . A totalTo verify the reliability of transcriptome analysis results, 39 DEGs were selected for qRT-PCR Figs. , 8 and 9PbPFP at the three stages was most consistent with the trend of lignin content, and the expression level of PbPFP was higher in the area with more stone cells (R) than in the area with fewer stone cells (L)  Fig. A-F. TherPbPFP overexpression vector was injected into ‘Dangshansuli’ fruits at 35 DAB. Compared with the uninjected sites, the content of lignin increased significantly at the injection sites of 35S-PbPFP, and the lignin content at the injection sites of pCAMBIA-1300 did not change significantly  leaves significantly increased after the transient expression of PbPFP as well  were up-regulated while one (Pbr008761.1) was down-regulated, and only one gene was identified as PbPFP (Pbr015118.1) and was up-regulated. The expression pattern of PbPFP at early fruit developmental stages in ‘Dangshansuli’ fruits was consistent with the trend of lignin content, which indicated that PbPFP might be involved in the biosynthesis of lignin in pear fruit. After transient expression of PbPFP in ‘Dangshansuli’ fruits at 35 DAB and tobacco leaves, the lignin content at the injection sites increased significantly, indicating that PbPFP might play a positive role in lignin biosynthesis in response to glucose treatment. It might be the result of regulating glycolysis and thus affecting carbon flux.Primary metabolism plays an important role in secondary metabolism . The inct sugars , which it sugars –36. The PbPAL1 (Pbr008363.1) and PbPAL2 (Pbr008387.1) increase the lignin biosynthesis in Arabidopsis stems [PbPAL1 responded to glucose stimulation in calli, and its expression was significantly up-regulated, while no significant changes was observed in PbPAL2. PbHCT49 (Pbr022422.1) and PbHCT65 (Pbr036245.1) were up-regulated by glucose. Among them, the expression pattern of PbHCT49 is consistent with the changes in stone cell content during pear fruit development [PbCOMT3 (Pbr040044.1) was significantly differentially expressed and up-regulated more than 6-fold in treated pear calli. In addition, another four PbCOMTs  were similarly up-regulated. PbPRX53 (Pbr026235.1) is closely related to the change of lignin content in pear fruits [PbPRX8 (Pbr000691.1) plays an important role in the early stage of fruit development [PbPRX8 and PbPRX55 were up-regulated by glucose, indicating that PbPRX8 and PbPRX55 might play an important role in the enhancement of lignin biosynthesis in response to glucose treatment.Glucose is an important signal molecule , 48, andis stems . PbPAL1 elopment . PbCOMT3r fruits . PbPRX8 ABA1, ABA3, ABI3, ABI5 and AAO3 [ABF genes  were up-regulated in response to glucose treatment  was up-regulated under glucose treatment, which might be one of the reasons for the increase of lignin content. The over-expressions of PvGA2ox5 and PvGA2ox9 in switchgrass reduced the lignin content of the transgenic lines, and the ratio of S/G lignin monomer was accompanied by a decrease in the expressions of lignin biosynthesis genes [GA2ox6, 4 of which were down-regulated, which might be related to the increase of lignin content in pear calli after glucose treatment  are involved in seedling development in response to glucose [AtbZIP1, as a sugar-sensitive TF, was negatively regulated by glucose [MYB103 results in a decrease in S-lignin content with an equivalent increase in G-lignin content [Pbr035306.1 and Pbr020777.1, as the homologous genes of AtMYB103, were down-regulated in the calli treated with glucose. It may be related to the fact that G-lignin is the main component of lignin in pear fruit [MYB52 could slightly induce the expression of genes in the secondary wall biosynthesis pathway [Pbr016851.1, Pbr002006.1 and Pbr035515.1, as the homologous genes of AtMYB52, were up-regulated in the calli treated with glucose. These genes might be involved in the glucose-mediated transcriptional regulation of the increase in lignin content in pear calli.The expressions of many TFs can also be induced by glucose. bidopsis , 63. OsM glucose , 65. Atb glucose . Some po content . Pbr0353ar fruit . MYB52 c pathway . Pbr0168PbPFP positively regulates lignin biosynthesis. Crosstalk of glucose signaling and hormone signaling, especially ABA, GA and SA signaling, and regulation of glucose-responsive TFs might play an important role in glucose-mediated lignin biosynthesis. This study is the first to use transcriptome analysis to investigate the regulatory role of glucose in lignin biosynthesis in stone cell formation in pear, providing a valuable resource for genetic research on the regulation of lignin biosynthesis. Future studies could focus on glucose-responsive transcription factors, and it would be very promising and meaningful to study how they sense glucose signals and regulate downstream phenylpropanoid pathway genes.Glucose metabolism is closely related to the phenylpropanoid pathway. However, the molecular basis of glucose regulating lignin biosynthesis in pear fruits and related genes has not been elucidated. In this study, exogenous glucose activated the glucose signaling pathway and modulated its downstream metabolic activities. 6566 DEGs were obtained by RNA-seq from glucose-treated pear calli. We have identified some candidate genes that might play important roles in glucose metabolism and lignin biosynthesis. Transient expression showed that P. communis) fruit were darkly grown on the MS solid medium with sucrose (30 g/L), 6-benzylaminopurine (0.5 mg/L), and 2, 4-dichlorophenoxyacetic acid (1.0 mg/L) according to Bai et al. [Pear calli derived from the flesh of young ‘Clapp’s Favorite  was cultivated under short-day conditions and the temperature was controlled at 25 °C.‘Dangshansuli’ fruits at 35 DAB were provided by Ziyinghe Orchard, Xiaji Town, Baoying County, Jiangsu Province, China. Tobacco  was taken to determine lignin content by the acetyl bromide method according to Tao . SamplesTotal RNAs were extracted using a Plant RNA Isolation Kit  according to the manufacturer’s instructions. The degradation of RNA was monitored on 1% agarose gels, and the purity of RNA was checked using a NanoPhotometer®spectrophotometer . RNA concentrations were measured using a Qubit®RNA Assay Kit in a Qubit® 2.0 Fluorometer . RNA integrity was evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system .Pyrus bretschneideri genome sequence (http://gigadb.org/dataset/100083).RNA-seq library preparation and sequencing were performed by the Beijing Allwegene Technology . RNA was enriched by magnetic beads with Oligo (dT). Then, the mRNA was broken into short fragments after the fragmentation buffer was added. Subsequently, six-base random primers (random hexamers) were used to reverse transcribe to synthesize one-strand cDNA using mRNA as a template, and then buffer, dNTPs and DNA polymerase I were added to synthesize two-strand cDNA. Next, AMPure XP beads were used to purify the double-stranded cDNA. The ends of the purified double-stranded cDNA were repaired, A and a linker were added. The double-stranded cDNA fragment size was selected by AMPure XP beads, and finally, PCR amplification was performed to construct a cDNA library. Then Agilent 2100 coupled with Illumina high-throughput sequencing platform (HiSeqTM2500/4000) was employed to check the library quality and sequence. The clean reads were mapped to the p-value [p-value < 0.05 and |log2(fold change)| ≥ 1.5 were selected. Based on the pear genome database, all differentially expressed genes were annotated through gene ontology  and Kyoto Encyclopedia of Genes and Genomes  [In order to determine the difference in gene expressions between the glucose treatment and the control calli, the gene expression level was calculated based on the fragments per kilobase per million (FPKM) corresponding to the clean-read counts. BH (fdr correction with Benjamini/Hochberg) method was used to control the false discovery rate to adjust the p-value , and thep/kegg/) . The GO http://plntfdb.bio.uni-potsdam.de/v3.0/), and then analyzed the annotated TFs with an e-value ≤10− 10 using BLASTX. Finally, annotated transcription factors from pears involved in response to the glucose treatment were analyzed and classified into different groups based on the standards of the PlnTFDB (version 3.0).We analyzed the DEGs determined by the annotated databases to mine for TFs using a Linux server. The data were further aligned with the protein sequences of the plant TF database PlantTFDB (version 3.0). Plant TFs were obtained from the TF database website (Tubulin (Pbr028019.1) was used as the reference gene, and the experiment was performed for three biological replicates. The PCR condition was 95 °C for 5 min, 55 cycles of 95 °C for 3 s, 60 °C for 10 s, 72 °C for 30 s, and the final extension was 72 °C for 3 min [-△△CT method [The coding region sequences of genes were downloaded from the pear genome, and Primer Premier 5 was used to design specific primers. The RNA samples used in the RNA-seq experiment were again used for qRT-PCR analysis. According to the manufacturer’s instructions, the first-strand cDNA was synthesized by one-step gDNA removal of transcripts and cDNA synthesis SuperMix (TRANGEN). The qRT-PCR used LightCycler480SYBR Green Master  system. The reaction system was LightCycler480SYBR green I Master  for 10 μL, each primer was 200 nM, template cDNA was 100 ng, double distilled water was added to 20 μl. or 3 min . After t Pbr02801.1 was usA. tumefaciens (GV3101), inoculated on the LB solid medium containing Knan and RFP, cultivate at 28 °C until a single colony grows, dip a sterile toothpick into the LB liquid medium, cultivated at 28 °C, centrifuge and resuspend the bacterial solution to the buffer , cultivate 3–6 h, use a needleless syringe to inject the bacteria containing the target gene vector into the pear fruits at 35 DAB and tobacco (Nicotiana tabacum L.) leaves. The small hole is close to the surface of the pear fruit, and the liquid is used to infiltrate the pear fruit with pressure to avoid mechanical damage. The fruits and tobacco are cultivated at 25 °C, first in the dark for 24 h, and then transferred to 16 h light, 8 h dark photoperiod for 5 d. Dying the pear longitudinally and determining the lignin content.According to Xue et al. , pCAMBIAp < 0.05, **p < 0.01, and ***p < 0.001).All data were statistically analyzed with Microsoft Excel  and SPSS 24 , and graphed with GraphPad Prism 5. There was a significant difference between the treatment and the control (*Additional file 1: Figure S1. Lignin content of calli treated with different concentrations of glucose. Different lowercase letters on the columns indicate that the treatment differs significantly at the 1% level.Additional file 2: Figure S2. Heat map of transcription factor expressions. A: ERF, B: bHLH, bZIP, TCP, C: C2H2, ARF, HSF.Additional file 3: Figure S3. Correlation analysis between RNA-Seq and qRT-PCR data.Additional file 4: Table S1. Sequencing data statistics Q20 analysis.Additional file 5: Table S2. Alignment of reads with reference sequence.Additional file 6: Table S3. The DEGs were identified as PbPFK/PFP.Additional file 7: Table S4. DEGs involved in plant hormone signal transduction pathways.Additional file 8: Table S5. The DEGs were identified as PbGA2ox6.Additional file 9: Table S6. Primers used for qRT-PCR analysis.


Were there regional differences in mortality rates and characteristics among decedents with hepatitis B listed as a cause of death in the US during 2000 to 2019?In this nationwide cross-sectional study including 35 280 decedents from 2000 to 2019, the highest hepatitis B–listed death rates were observed in coastal and Appalachian states; in addition, younger median age at death occurred predominantly in Appalachian states. Most decedents, regardless of birthplace, had liver-related conditions listed as underlying cause of death, and decedents born in the US, who constituted approximately two-thirds of all deaths, more frequently had nonliver conditions listed as underlying cause of death compared with non-US–born decedents.These findings suggest that in addition to addressing liver-related complications, US-born persons with chronic infection may also require diagnosis and management of multiple comorbidities. This cross-sectional study assesses characteristics of US decedents with hepatitis B–listed deaths during 2010 to 2019 and compares rates with 2000 to 2009. US hepatitis B mortality has been described nationally, but examination subnationally may identify differences in mortality rates and decedent characteristics, including birthplace.To examine characteristics of decedents with hepatitis B–listed deaths during 2010 to 2019 and compare age-adjusted hepatitis B–listed death rates during 2010 to 2019 vs 2000 to 2009.This cross-sectional study used Multiple Cause of Death data from 50 US states and the District of Columbia (DC) from 2000 to 2019 to assess characteristics of US residents with hepatitis B listed as an underlying cause of death (UCOD) or contributing cause of death on death certificates. Data were analyzed from September 2019 to May 2022.Hepatitis B listed as underlying or contributing cause of death.Outcomes of interest were hepatitis B–listed death counts, age-adjusted rates, and characteristics of decedents during 2000 to 2019. The distribution of hepatitis B–listed deaths according to sociodemographic characteristics and UCOD among US- and non-US–born decedents were also examined.P < .001). Hepatitis B was the UCOD among approximately 30% of US- and non-US–born decedents with hepatitis B COD. Irrespective of birthplace, most decedents had liver-related UCOD. Compared with non-US–born decedents, US-born decedents more frequently had nonliver conditions listed as UCOD. Liver cancer was the predominant UCOD among non-US–born decedents (37.9% of decedents). From 2000 to 2009 compared with 2010 to 2019, the hepatitis B–listed mortality rate significantly decreased nationally  and in 14 states; significant increases were observed in West Virginia  and Kentucky .A total of 35 280 decedents with hepatitis B listed as the cause of death were identified, including 17 483 deaths during 2010 to 2019. Decedents were 63.3% US-born, and 25.8% of decedents were Asian or Pacific Islander and 46.5% of decedents were White; 28.4% of decedents were listed as having hepatitis C virus (HCV) or HIV coinfection. State-level rates significantly surpassed the overall US rate (0.47 deaths per 100 000 population) in DC , Hawaii, Oklahoma, California, Tennessee, West Virginia, Mississippi, Oregon, Washington, Louisiana, Kentucky, and New York . Median (IQR) age at hepatitis B death was significantly younger in Kentucky (54.0 [46.0-64.0] years), West Virginia (56.0 [47.0-65.0] years), Tennessee (57.0 [50.0-65.0] years), Mississippi (58.0 [50.0-65.0] years), and Ohio (59.0 [50.0-66.0] years) than the national median (60.0 [53.0-69.0] years), which itself was significantly younger than nonhepatitis B–listed deaths (77 [63.0-87.0] years; These findings suggest that US-born decedents constituted two-thirds of all hepatitis B–listed deaths and median age at death was youngest in Appalachian states. Irrespective of birthplace, most decedents had liver-related UCOD; however, US-born decedents more frequently had nonliver UCOD than non-US–born decedents. In addition to addressing liver-related complications, US-born persons with chronic infection may also require diagnosis and management of multiple comorbidities. In addition to increased mortality from liver-related causes, chronic hepatitis B has been associated with premature mortality and elevated mortality rates from all causes.13 Annual national viral hepatitis surveillance reports have included hepatitis B mortality estimates since 2004, displaying state-level data since 2015.13 However, these reports lacked information on decedent place of birth, comorbidities, and underlying vs contributing causes of death. In 2019, states with the highest hepatitis B–listed death rates included Hawaii, Oklahoma, Tennessee, and Oregon. US Multiple Cause of Death (MCOD) data are compiled from all US-registered deaths that are housed in each state’s vital registration office. Demographic information, such as state of residence, US- vs non-US birthplace, age, race and ethnicity, and sex are available in MCOD data and are highly complete.National hepatitis B death rates were relatively constant during 1999 to 2019 and highest among decedents aged 55 years and older, non-Hispanic Asian and Pacific Islander persons, and men.14 The plan described core indicators to measure progress, including a 65% reduction in the rate of hepatitis B deaths by 2030. One specific goal of the plan was to “improve public health surveillance through data collection, case reporting, and investigation at the national, state, tribal, local, and territorial health department levels.”14 State and local health departments were encouraged to develop chronic hepatitis B surveillance programs, including understanding hepatitis B mortality in their jurisdiction, and to prioritize activities according to the needs of the populations they serve.In 2021, the Department of Health and Human Services (DHHS) published a national strategy for eliminating hepatitis B as a public health threat in the United States.16 In the most recent analysis of the National Health and Nutrition Examination Survey, persons of Asian descent constituted 69.7% of chronic hepatitis B cases.15 Compared with US-born persons, non-US–born persons typically acquire infection earlier in life, which can affect clinical outcomes, including a 25% lifetime risk of premature mortality from liver failure or liver cancer.19 We surmised that, compared with US-born decedents, more non-US–born decedents would have hepatitis B listed as the underlying COD (UCOD). To further explore differences between US- and non-US–born decedents with hepatitis B–listed deaths, we examined the distribution of these deaths by sociodemographic characteristics, median age at death, and UCOD.Accordingly, we examined MCOD data for decedents with hepatitis B listed as an underlying or contributing COD during 2000 to 2019 to understand subnational variability in death counts and rates, characteristics of decedents , and changes in death rates. Recent US hepatitis B prevalence estimates reported higher numbers and rates of chronic hepatitis B among non-US–born persons compared with those born in the US.STROBE) reporting guideline.This cross-sectional study was deemed exempt from institutional review board review and informed consent because all data were obtained from secondary sources without personally identifiable information, per HHS Code of Federal Regulations Title 45 Part 46 2018. This study was reported according to the Strengthening the Reporting of Observational Studies in Epidemiology  research review committee to obtain information on decedent state of residence and US birthplace status.21Region- and state-level hepatitis B–listed deaths included decedents whose residence was in the 50 US states and the District of Columbia (DC). Regions were grouped according to the assignments under the 10 DHHS regional offices.International Statistical Classification of Diseases and Related Health Problems, Tenth Revision (ICD-10)22 COD codes if at least 1 code indicative of hepatitis B  was listed as the UCOD or a contributing COD (CCOD) in the record axis fields. We captured the UCODs and all CCODs to ensure completeness.24 If a decedent had hepatitis B listed as a COD, we looked for evidence of coinfection with HCV (B17.1 and B18.2), HIV (B20-B24), and HDV  present as any COD. A US-born decedent was defined as a birth in the 50 states or DC. A non-US–born decedent was defined as a birth in any country outside of the United States.We defined hepatitis B–listed deaths through 25 To examine variability in rates during 2010 to 2019, we derived death rate ratios (DRRs) to compare hepatitis B–listed death rates in DHHS regions and states to that of the United States as a whole, denoted by DRR = (RegionRate or StateRate)/USRate). Z-score P values were examined to determine if differences in death rates were statistically significant for each region and state compared with the United States and 2010 to 2019 compared with 2000 to 2009. We were unable to calculate hepatitis B–listed death rates by US birthplace status owing to lack of such information in the US Census population data sets. Race and ethnicity information is reported by the funeral director by questioning the decedent’s next of kin or direct observation .26 Race and ethnicity data were included to describe race and ethnicity disparities by overall, US birthplace status, and coinfection status.Hepatitis B–listed death counts were enumerated by aggregating data into two 10-year time intervals (2000-2009 and 2010-2019) to improve reliability of state-level measures. Hepatitis B–listed death rates in 2000 to 2009 and 2010 to 2019 were calculated by dividing the number of hepatitis B–listed deaths in a state of residence and time period by the equivalent Census population. Death rates were adjusted to the age distribution of the 2000 US standard population using the direct method. To estimate the variance of age-adjusted death rates, 95% CIs were calculated based on a γ distribution, as developed by Fay and Feuer.2 test and 95% CIs of proportions were used to determine significant differences in the proportion of hepatitis B–listed deaths among US-born and non-US–born individuals and who had HCV, HIV, or HDV coinfection listed and did not have coinfection listed for each state relative to that proportion of hepatitis B–listed deaths for the United States.Median age at hepatitis B–listed death, distribution of the status of US birthplace, and HCV, HIV, or HDV coinfection during 2010 to 2019 were examined by state. The Kruskal-Wallis test was used to assess statistically significant differences in median age at hepatitis B–listed death for each region and state relative to the median age at hepatitis B–listed death for the United States. The Pearson χWe determined whether there were differences according to US vs non-US birth status by examining the distribution and median age of hepatitis B–listed deaths by sociodemographic and UCOD categories during 2010 to 2019. Among decedents for whom hepatitis B was not listed as the UCOD , we classified the UCOD into 15 broader categories  and the map was generated using R Studio, version 1.3.1056 . Data were analyzed from September 2019 to May 2022.We followed NCHS’s policy regarding decedent confidentiality; therefore, we did not display or interpret any subnational results where counts were fewer than 10 deaths. All mortality rates are expressed per 100 000 population. To control for type I errors arising from multiple comparisons, we adjusted From 2000 to 2019, there were 35 280 decedents with hepatitis B listed as the cause of death, including 17 797 deaths from 2000 to 2009  and 17 483 deaths from 2010 to 2019  . HepatitThe number of hepatitis B–listed deaths in 13 states exceeded the national mean number of all hepatitis B–listed deaths (mean [SD] 343 [550] deaths per state) . CaliforStates in which the hepatitis B–listed death rates significantly surpassed the national hepatitis B–listed death rate included DC , Hawaii, Oklahoma, California, Tennessee, West Virginia, Mississippi, Oregon, Washington, Louisiana, Kentucky, and New York  . The higThe median (IQR) age at hepatitis B–listed death was 60.0 (53.0-69.0) years for the United States . SignifiAmong hepatitis B–listed deaths, US-born decedents, compared with decedents not born in the US, were more frequently aged 45 to 64 years , born during 1945 to 1965 , non-Hispanic Black , non-Hispanic White , and listed with HCV, HIV, or HDV coinfection  . In 21 sDuring 2010 to 2019, 4969 hepatitis B–listed deaths (28.6%) were also listed with HCV, HIV, or HDV coinfection (only 6 deaths listed HDV infection). Hepatitis B–listed deaths with HCV, HIV, or HDV coinfection, compared with hepatitis B–listed deaths without coinfection, were more frequently aged 45 to 64 years , born during 1945 to 1965 , Hispanic , non-Hispanic Black , non-Hispanic White , and US-born . The proportion of coinfections among hepatitis B–listed deaths in 11 states significantly exceeded the national distribution (28.4%); 41.0% of these deaths occurred in Ohio, Kentucky, Pennsylvania, Tennessee, and West Virginia  (P < .001)  . Among UP = .24) . Among d < .001) . In cont < .001) . Compare < .001) . Irrespe < .001) .P < .001) , significant increases were observed in West Virginia , and Kentucky   . Althoug = .005) .28 In all, areas with significantly higher hepatitis B–listed death rates than the national hepatitis B–listed death rate were primarily in the coastal and Appalachian regions. Compared with the national median age at hepatitis B–listed death, significantly younger median age at death occurred among decedents of 5 Appalachian states, where most decedents were US-born and approximately one-third had HCV or HIV coinfection. In contrast, a significantly older median age at death was observed among California decedents, who were predominantly not born in the US. Hepatitis B–listed deaths for persons not born in the US occurred primarily in West and East Coast states with large immigrant populations from hyperendemic areas.29 Non-US–born decedents more frequently had an older median age at death compared with the national median age at hepatitis B–listed death.In this cross-sectional study assessing hepatitis B–listed deaths in the US, we observed a hepatitis B–listed death rate of 0.47 deaths per 100 000 population during 2010 to 2019, which represented significant declines for the US as a whole and for 14 states compared with 2000 to 2009. However, significant increases in hepatitis B–listed death rates were observed in West Virginia and Kentucky. These states also experienced the highest death rates from all causes in 2017.30 it is likely that HDV coinfection deaths have been continually underreported.Nearly two-thirds of decedents with hepatitis B–listed deaths were born in the US; in 21 states and DC, the fraction of US-born decedents exceeded the national distribution. US-born decedents were more frequently coinfected with HCV or HIV than those not born in the US. During 2010 to 2019, only 6 decedents had HDV coinfection listed. Given that HDV testing is conducted inconsistently in the United States,31 found that since 2014, these conditions also contributed to decreases in the overall US life expectancy, especially in areas where we observed high proportions of US-born hepatitis B–listed deaths and younger median ages at death, such as the Ohio River Valley and Appalachian region.We found no difference in the frequency with which hepatitis B was listed as the UCOD among US- and non-US–born decedents. However, when hepatitis B was listed instead as a CCOD, decedents not born in the US more often had liver cancer listed as the UCOD, whereas US-born decedents more often had UCOD conditions that included HCV infection, other viral hepatitis, HIV infection, alcoholic liver disease, respiratory and circulatory conditions, and drug overdose, alcohol poisoning, suicide, or homicide. A recent vital statistics analysis32 Whether infected at birth or later in life, all persons with chronic hepatitis B need continual, lifelong medical care to monitor disease activity to determine whether and when antiviral treatment is indicated, and to undergo periodic surveillance for hepatocellular carcinoma as recommended.32 Irrespective of birthplace status or whether hepatitis B was listed as the UCOD or a CCOD, we found that decedents with hepatitis B COD had a significantly younger median age at death than decedents without hepatitis B listed as a COD. Most decedents with hepatitis B listed as a COD, regardless of birth status, had liver-related UCOD.As chronic hepatitis B is a heterogeneous condition with a highly variable and unpredictable course, all persons with chronic infection require continual lifelong assessment, as a sizable proportion at some point may be indicated for hepatitis B antiviral therapy.24 Studies using medical records to validate death certificate COD have consistently shown that mortality data greatly underestimates the true hepatitis B mortality burden, even when persons with hepatitis B infection had a liver-related COD.33 Although findings from this study may be underestimated, they nonetheless support the consideration of CCOD in addition to the UCOD, and, more importantly, attest to its capacity to permit identification of the various conditions—especially those leading to early death—afflicting persons with chronic hepatitis B in the United States.24 Next, we were not able to calculate national age-adjusted hepatitis B–listed death rates in the US-born and non-US–born populations because those population-specific denominators are not available in the US Census population data. Additionally, American Indian and Alaska Native persons are often misclassified as other racial and ethnic groups on death certificates,26 resulting in underreporting of conditions among American Indian and Alaska Native persons. Furthermore, geographic characterizations among non-US–born persons should be interpreted with caution, as they may change their residence often before settling.This study has some limitations. Since this analysis hinged exclusively on the interpretation of MCOD data, its principal limitations were the use of data based on clinician judgment about causes and relative importance of conditions leading to death. Death certificates have been shown to have high rates of error, particularly as they pertain to chronic liver disease.The findings of this cross-sectional study suggest that in addition to diagnosis and management of hepatitis B among non-US–born persons, US-born persons with chronic infection, who constituted approximately two-thirds of all hepatitis B–listed deaths, may also require diagnosis and management of viral coinfections, respiratory and cardiovascular conditions, nonviral liver disease, and addiction-related sequelae. Further reduction of hepatitis B mortality will require access to hepatitis B-related care that is continual and life-long. Universal hepatitis B screening in adults is expected to identify more persons with hepatitis B in need of linkage to comprehensive and routine medical care.


Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease affecting the spine and sacroiliac joints. To investigate whether there are differences in inflammatory and chronic structural damages, as assessed by a semiquantitative MRI scoring method, between non-radiographic axial spondyloarthritis (nr-axSpA) and ankylosing spondylitis (AS) patients with active inflammation at baseline, and to evaluate the treatment response in these patients after 3 months of tumor necrosis factor-alpha (TNF-α) inhibitor treatment.Fifty-eight axSpA patients with active inflammation were included in the study. The patients were divided into nr-axSpA group and AS group. MRI examinations of the sacroiliac joints were performed before and after treatment. Inflammatory and structural damages in these patients were assessed using the established Spondyloarthritis Research Consortium of Canada (SPARCC) inflammation and sacroiliac joint structural (SSS) scoring methods, which are two MRI-based scoring methods. The SPARCC score, SSS score, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) level were compared between the two groups.P > 0.05); however, SSS scores for fat metaplasia, erosion, and backfill for patients in the AS group were significantly higher (P < 0.001). Compared with baseline, SPARCC scores were significantly decreased in both groups after treatment (P < 0.001); however, after treatment, no statistically significant difference was found regarding SPARCC scores between the AS and nr-axSpA groups. Compared with baseline, a significant increase in the SSS scores for fat metaplasia and backfill (P < 0.001) and a significant decrease in the SSS scores for erosion (P < 0.001) were observed in all axSpA patients. Changes in the SPARCC score was inversely correlated with the changes in the SSS score for fat metaplasia . Changes in the SSS score for backfill were positively correlated with the changes in the SSS score for fat metaplasia  and inversely correlated with those for erosion .At baseline, SPARCC scores for patients in the nr-axSpA and AS groups did not differ significantly (The SPARCC and SSS scoring systems can be used to assess inflammatory and chronic structural damages as well as treatment responses in patients with axSpA. More severe structural damages were seen in AS patients. TNF-α inhibitor treatment for 3 months could effectively reduce inflammation in axSpA patients.The online version contains supplementary material available at 10.1186/s12891-022-05609-x. Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease affecting the spine and sacroiliac joints. It can be categorized as non-radiographic axial spondyloarthritis (nr-axSpA) and radiographic axial spondyloarthritis (r-axSpA); the latter is also termed ankylosing spondylitis (AS) . AlthougMRI, a noninvasive and sensitive technique, is an established method for diagnosing and managing axSpA . InflammSystematic MRI-based scoring systems for inflammatory and structural damages in the sacroiliac joints have been developed and validated . The SpoThis study assessed the differences in the inflammatory and structural damages displayed on MRI between nr-axSpA and AS patients with active inflammation at baseline; furthermore, it investigated the response of axSpA patients to tumor necrosis factor-alpha (TNF-α) inhibitor treatment using a semiquantitative MRI-based scoring system.This study was a longitudinal cohort study consisting of patients aged 18–45 years who had symptoms for more than 3 months but less than 5 years. Patients were included if they were diagnosed with axSpA and had active inflammation on MRI. These patients were diagnosed according to the 2009 Assessment of SpondyloArthritis International Society (ASAS) criteria (either nr-axSpA or AS if patients had sacroiliitis on X-ray meeting the Modified New York Criteria) , 21.In total, 58 patients with axSpA who had baseline and 3-month after-treatment  scans were evaluated systematically according to a standardized protocol. Because of the study’s retrospective nature, flexible windows of ±1 month for MRI scans were permitted. These patients were assessed to be suitable for TNF-α inhibitor treatment by physician and had not previously been treated with other biological agent. For TNF-α inhibitor treatments, patients received either etanercept or adalimumab. This study was approved by the Institutional Review Board of Tianjin First Central Hospital Medical Ethics Committee (2021N143KY), and the requirement of informed consent was waived because the study was retrospective. All experiments were conducted in accordance with the Declaration of Helsinki (1964), and all methods were performed following the relevant guidelines and regulations.All patients were scanned using a 3.0 Tesla (T) MR scanner . All sequences were obtained by scanning the oblique coronal plane parallel to the sacrum plane. The scanning sequences and parameters were as follows: modified Dixon turbo spin echo (mDixon TSE) T1-weighted images (T1WIs) (time of repetition (TR), 462 ms; echo time (TE), 16 ms; field of view (FOV), 220 × 220 mm; slice thickness, 3 mm; slice gap, 0 mm; matrix, 276 × 210), mDixon-TSE T2-weighted images (T2WIs) . After scanning, T1WIs and T2WIs were automatically reconstructed and generated.BME appears as the low-intensity signal on T1WIs and high-intensity signal on T2WIs and is clearly present in the subchondral bone marrow .Structural damages include bone erosion, fat metaplasia, backfill, and bone bridges/ankylosis . All defErosion: full-thickness loss of the dark appearance of cortical bone and loss of the normal appearance of the adjacent bone marrow.Fat metaplasia: the signal is brighter than normal bone marrow, which meets the following conditions: homogeneous; located in the subchondral bone; sharply defined borders.Backfill: increased signal at a complete loss of the cortical bone, clearly demarcated from the adjacent normal marrow by an irregular dark signal.Ankylosis: bone marrow signal extending between the sacral and iliac bone marrow with full-thickness loss of the dark appearance of the iliac and sacral cortical bone.All MRIs of the sacroiliac joints were evaluated for BME according to the SPARCC method . First, All MRI images of the sacroiliac joints were evaluated for fat metaplasia, erosion, backfill, and ankylosis according to the SPARCC SSS method, which was used to assess the structural lesions on T1WIs . The preReaders were blinded to patient demographics and treatments. A calibration exercise was conducted in 10 cases randomly selected from the cohort using baseline and 3-month MR scans after TNF-α inhibitor treatment scored by two radiologists blinded to the time points.Baseline and 3-month MR scans of the sacroiliac joint were scored independently by two radiologists, who were blinded to all clinical data.Spearman correlation analysis. Interobserver reliability for baseline and 3-month scores was assessed using the intraclass correlation coefficient (ICC). The correlation strengths were defined as follows: 0–0.2, poor; 0.3–0.4, fair; 0.5–0.6, moderate; 0.7–0.8, strong; > 0.8, excellent agreement. P < 0.05 was considered statistically significant.All data were statistically analyzed using the SPSS 26.0 software. Continuous data are expressed as means ± standard deviation (SD), and categorical data are expressed as percentages. Analyses were performed using the mean scores from the two readers. Normally distributed variables were compared using an independent t-test, and non-normally distributed variables were compared using the Mann-Whitney U test. Categorical variables were compared between groups using the chi-squared test. Associations between changes in parameters of inflammation (CRP and SPARCC score) and SSS subgroup scores were examined using the A total of 58 patients were evaluated. The study flow diagram is presented in Fig. At baseline, 38 patients (65.52%) had BME on both sides; 33 patients (56.90%) had BME with a depth greater than 1 cm. Almost all patients (93.1%) had BME on the sacral side of the sacroiliac joints, either unilateral or bilateral. Fat metaplasia was found in 51 patients (87.93%), of which 41 (80.39%) were bilaterally affected. Erosion was found in 53 patients (91.38%), and backfill was found in only 22 patients (37.93%).The mean ± SD SPARCC score was 24.79 ± 17.13. Most patients had a score of > 0 for fat metaplasia, erosion, and backfill in the sacroiliac joints. No patients showed ankylosis at baseline. The mean ± SD SSS was 26.93 ± 20.26. A higher SPARCC score was observed in the AS group than in the nr-axSpA group; however, this difference was not significant. Significant differences were found in the SSS for fat metaplasia, erosion, and backfill at baseline  (Table P < 0.001) (Table P < 0.001) and a decrease in SSS for erosion (P < 0.001) were observed in axSpA patients after treatment. The variations in the SPARCC scores for BME and the SSS scores for fat metaplasia, erosion, and backfill in axSpA patients, as well as the difference between the nr-axSpA and AS groups, are shown in Figs. Compared with baseline, SPARCC scores were significantly decreased after treatment in the AS group (1) Table . Similar1) Table . Howeverr = − 0.634, P < 0.001) and backfill . The changes in the SSS score for backfill were positively correlated with the changes in fat metaplasia  and inversely with the changes in erosion  . A strong to excellent interobserver reliability was achieved for the identification of fat metaplasia at the 3-month follow-up (ICC 0.75–0.91).This semiquantitative analysis analyzed the inflammatory and structural damages based on MRI images of axSpA patients at baseline and after 3 months of treatment, which demonstrated that MRI SPARCC scoring system could be used to evaluate the inflammation and therapeutic efficacy in patients with axSpA. The results indicated that patients with AS had more severe structural damage, and TNF-α inhibitor treatment could effectively reduce inflammation in axSpA patients after 3 months.At baseline, almost all patients had either unilateral or bilateral BME on the sacral side of the sacroiliac joints. More than half of the patients had BME that extended more than 1 cm from the sacroiliac joint space, a condition known as deep inflammatory lesions. Different levels of BME showed different courses of development during the follow-up period. This demonstrates the need for a more accurate method to assess BME, and the SPARCC scoring system, which can be used to assess disease activity, has been proposed as a suitable method –25. In tMRI data from prospective observation suggest that the development of new tissue follows the resolution of inflammation in the erosion cavity. This phenomenon, termed “backfill” , 28, hasSeveral previous studies have found that fat metaplasia and backfill are key intermediary steps in the development of new bone formation , 14, butThis study has several limitations. First, the sample size is small; however, despite the relatively limited sample, this work offers valuable insights. Second, because of its retrospective nature, the timing of MRI examinations was not similar in all patients. Third, the study could not analyze the prognostic implications of new bone formation. Considering these limitations, larger studies performed over a longer follow-up period should be conducted.In conclusion, MRI-based scoring methods such as the SPARCC and SSS can be used to assess inflammatory and chronic structural damages in axSpA as well as patients’ responses to treatment. More severe structural damage was observed in AS patients with active inflammation at baseline. TNF-α inhibitor treatment effectively reduced inflammation in axSpA patients after 3 months.Additional file 1.Additional file 2.Additional file 3.


The median overall survival (OS) was 45, 44 and 20 months for patients without Ps, PPs and EMPs, respectively (p = 0.013). Patients with PPs who underwent ASCT had similar OS than those without Ps (98 vs. 113 months) and significantly longer than those with EMPs . In patients non-eligible for ASCT the presence of PPs or EMPs was associated with shorter OS compared with patients without Ps . In the relapsed setting, a significant survival benefit was observed beyond the year 2000, but still with significant differences among patients without Ps, PPs and EMPs . Importantly, rescue therapy with combinations of proteasome-inhibitors plus immunomodulatory drugs was associated with prolonged OS from first relapse (over 6 years), even in patients with EMPs.From January 1970 to December 2018, 1304 patients were diagnosed with multiple myeloma (MM) at our institution and 256 (19.6%) had plasmacytomas (Ps) . Patients with Ps had lower serum M-protein and less advanced ISS stage than those without. At first relapse, 192 out of 967 patients (19.8%) developed Ps . The only factor associated with Ps at relapse was the presence of Ps at diagnosis (46% vs 13%,  Multiple myeloma (MM) is a proliferation of clonal plasma cells that produce a monoclonal protein detectable in serum and/or urine and is characterized by the presence of CRAB manifestations . Although myeloma plasma cells have a strong dependence of the bone marrow microenvironment, in up to one-third of patients the plasma cell proliferation escape the microenvironment influences resulting in soft-tissue plasmacytomas –4. In faThe presence of plasmacytomas is considered an adverse prognostic factor, being worse for EMPs in comparison with PPs , 9–11. AIn this context, the aim of our study was to analyze the incidence, location and outcome of patients with MM and plasmacytomas diagnosed at our institution over a long period of time, focusing on the two types of plasmacytomas: PPs and EMPsAll patients with MM diagnosed and treated at Hospital Clínic from Barcelona  between January 1970 and December 2018 were included in the study. Patients with primary plasma cell leukemia or solitary plasmacytoma were excluded. Baseline characteristics, treatment and follow-up data were obtained from our database continuosly updated. Plasmacytomas were assessed by physical examination and/or imaging methods and histologically confirmed whenever consider necessary. Plasmacytomas arising from a lytic bone lesion were classified as PPs, while plasmacytomas with no contact with bone were considered EMPs. Patients presenting simultaneously with both, PPs and EMPs, were considered as EMPs for the survival analysis. Medical records and radiological studies were accurately reviewed to determine the locations and type of plasmacytomas. All data were reviewed by at least one senior author of the paper (JB or LR). Cytogenetic data were available in a minority of patients for historical reasons and were not considered in the analysis. The International Staging System (ISS) was retrospectively applied in all patients with albumin and β2-microglobulin measurements at diagnosis. Patients were categorized into two calendar periods according to the availability of novel agents: 1970-1999 (period 1) and 2000-2018 (period 2). Therapeutic regimens were grouped in 5 categories: conventional chemotherapy-based, proteasome inhibitor-based (PI), immunomodulatory-based (IMiD), proteasome inhibitor plus immunomodulatory drug based (PI + IMiD) and monoclonal antibody-based (MAb). Response to therapy and progression were assessed according to Uniform Response Criteria for MM . This st2 or Fisher’s exact test was used when required to assess the statistical significance of multiple comparisons. Overall survival (OS) was calculated from the date of diagnosis to death or last follow-up for censored cases. Survival curves were plotted according to the Kaplan and Meier method and statistically compared by means of the log-rank test.Categorical variables were described as frequency and percentage and continuous variables were reported as mean and standard deviation (SD) or median and range. The χAll analyses and graphs were obtained using the statistical software IBM SPSS version 25.p = 0.03). Patients with plasmacytomas at diagnosis had a higher percentage of Bence-Jones  and oligosecretory myeloma , as well as a less advanced ISS stage . Of note, the serum M-protein size  and bone marrow infiltratrion  was significantly lower in patients with PPs compared with patients without plasmacytomas.A total of 1304 patients were included in the analysis and 256 of them (19.6%) had plasmacytomas at diagnosis. Patient’s baseline characteristics are summarized in Table The incidence of the two types of plasmacytomas at diagnosis was 17.6% (230 patients) and 1.9% (26 patients) for PPs and EMPs, respectively. There were not significant differences in baseline characteristics between patients with both subtypes of plasmacytomas  than for the EMPs group (1.5% to 2.3%) , paravertebral (39.1%), skull (13%) and pelvis (11.3%). In the EMPs group the most commonly involved sites were pleura (23%), skin (19.2%) and liver (15.3%). Central nervous system (CNS) involvement at diagnosis was extremely uncommon with only one patient. Location of plasmacytomas is depicted in Table The initial treatment evolved overtime. Thus, in the overall series, all patients diagnosed before the year 2000 were treated with chemotherapy while after the year 2000, 42.3% received chemotherapy, 24.7% PI, 9.6% IMiDs, 20.6% PI + IMiD and 2.1% MAb. Seventy-seven (13.3%) patients received an autologous stem cell transplant (ASCT) in period 1 compared with 336 (46.2%) patients in period 2. The induction treatment in each subgroup of patients according to the type of plasmacytomas is summarized in Supplementary Table p = 0.013)   and had a significantly longer OS than patients with EMPs . In contrast, in patients non-transplant eligible the presence of EMD, either PPs or EMPs, confers worse prognosis compared with patients without plasmacytomas, being dismal for those with EMPs  . There was a trend towards a shorter OS for patients with EMD when treated with IMiD therapy  and PI + IMiD therapy . Patients who received a initial PI regimen had a similar OS regardless the presence of extramedullary involvement, either PPs or EMPs   were alive at the time of this analysis with a median follow-up in survivors of 82 months. The median OS was 45, 44 and 20 months for patients without plasmacytomas at diagnosis, patients with PPs and patients with EMPs, respectively (13) Fig. . Althoug86) Fig. . When wely) Fig. . Finallyp = 0.0046). No differences in the incidence of plasmacytomas at first relapse were observed between periods 1 and 2 (Table p < 0.00001). The incidence of plamacytomas at relapse in patients who underwent up-front ASCT vs. those who did not was 22% vs 17.8% (p = 0.22). No differences in the incidence of plasmacytomas were observed between patients initially treated with chemotherapy or new drugs . Fifty six percent of patients relapsing with plasmacytomas had involvement in two or more locations. The location of PPs were similar to that observed at diagnosis . Regarding EMPs, we observed an increased incidence of skin involvement (40%), while the incidence in other locations was quite similar to that observed at diagnosis . CNS involvement was also uncommon with only 4 patients, representing the 2% of the overall series  patients relapsed. Data regarding the presence, type and location of plasmacytomas were available in 967 of the 991 patients. One hundred ninety two (19.8%) patients developed plasmacytomas at first relapse. The incidence of PPs at first relapse was similar (14.6%) to that observed at diagnosis while the incidence of EMPs significantly increased at relapse .Treatment of patients at first relapse is summarized in Table p = 0.116)  for patients without plasmacytomas, patients with PPs or patients with EMPs  . PI or IMiD-based regimens improved the outcome in all subgroups, although the benefit was lower for patients with plasmacytomas. Thus, in patients receiving a PI-based regimen the OS was 53, 22 and 16 months (p = 0.0001) for patients without plasmacytomas, with PPs and with EMPs, respectively. In patients receiving an IMiD-based regimen the OS was 49, 34 and 25 months (p = 0.117), respectively. Salvage therapy with a PI + IMiD based-regimen substantially improved the median OS in all subgroups of patients with no statistically significant difference among them: 73 vs. not reached vs. 90 months (p = 0.414) for patients without plasmacytomas, patients with PPs and patients with EMPs, respectively  Fig. . We compMPs Fig. . In patily) Fig. . The OS This study describes the incidence, presenting features and impact on survival of EMD in a large cohort of patients diagnosed with MM and followed at an academic institution over a long period of time, from 1970 to 2018. According to the consensus on EMD definition recently published , the outp = 0.01) [p = 0.78 and 3-yr OS 80.1% vs. 77.7%, p = 0.09). In contrast, patients with EMPs had a significantly worse 3-yr PFS of 39.9% in comparison to patients without plasmacytomas (p = 0.001) and PPs patients (p = 0.007), and a significantly worse 3-yr OS of 58% compared to patients without plasmacytomas and patients with PPs (p < 0.001 in both cases) [p = 0.034) and the median OS was 46.5 vs. not reached (p = 0.002) for the EMPs and PPs groups, respectively [P = 0.086 for patients without plasmacytomas, patients with PPs and patients with EMPs, respectively). As reported by others, ASCT overcomes the bad prognosis of PPs, but not EMPs [Extramedullary disease is generally considered a poor prognostic feature, but few reports discriminate between the two types of plasmacytomas. Wu et al. reported a shorter survival for patients with de novo MM and plasmacytomas treated with conventional chemotherapy compared with patients without plasmacytomas. However, patients who underwent ASCT had similar outcome, irrespective of the presence or absence of plasmacytomas . Vareton = 0.01) . This is = 0.01) . Two stu = 0.01) , 29. A lh cases) . In a renot EMPs , 29. Thup = 0.006) [p = 0.006) [p = 0.093) [In the relapse setting, Pour et al. reported that soft-tissue involvement was associated with a poor prognosis, particularly in patients with EMPs compared with PPs  . Mangiac= 0.006) . More re= 0.093) . Accordi= 0.093) , 32 and = 0.093)  have sho= 0.093) . With th= 0.093) . In a re= 0.093) . There a= 0.093) , 38. Thu= 0.093) . However= 0.093) , 40. Thi= 0.093)  will hopIn summary, patients with plasmacytomas have less tumor burden than those without plasmacytomas. It is important to distinguish between the two types of plasmacytomas, as they display different outcomes. EMPs are more frequent at relapse than at diagnosis while the incidence of PPs is similar at diagnosis and at relapse. The only factor associated with the development of plasmacytomas at relapse is the presence of plasmacytomas at diagnosis. Patients with PPs at diagnosis undergoing high-dose therapy have similar survival than those without plasmacytomas while patients with EMPs had poorer outcome than those with PPs or those without soft-tissue involvement. At relapse, there are still significant differences in survival among patients without plasmacytomas, with PPs and with EMPs, despite the improvement in outcomes observed in all subgroups after the introduction of novel drugs. Prospective analysis focused on survival outcomes in patients with MM and clearly defined PPs and EMPs with the new wave of antimyeloma approaches should be encouraged.Supplementary tables and figures


Vocal cord paralysis caused by recurrent laryngeal nerve (RLN) injury during thyroidectomy results in hoarseness, aspiration, and dyspnea. We evaluated the usefulness of nerve guidance conduits (NGCs) constructed from an asymmetric polycaprolactone (PCL)/Pluronic F127 porous membrane and filled with platelet-rich plasma (PRP) for functional RLN regeneration. We evaluated the proliferation and migration of Schwann cells (SCs) after PRP treatment in vitro. For the in vivo study, rabbits were divided into a non-loaded NGC group and a PRP-loaded NGC group. The left RLNs were resected and interposed with the NGCs. Functional and histological examinations of the vocal cords were performed. SC proliferation and migration increased in a PRP dose-dependent manner, with the PRP increasing the levels of neurotrophic factors, myelin-associated glycoprotein, and ERK. In vivo, the PRP group showed significantly better vocal cord mobility and less vocalis muscle atrophy than the non-loaded NGC group. Histologically, the ingrowth of nerve endings occurred more rapidly in the PRP group, and acetylcholinesterase, neurofilament, and S-100 expression in neural endings were significantly higher in the PRP group. Furthermore, transmission electron microscopy showed that myelinated axons were more tightly packed in the PRP group. This study shows that PRP-loaded NGCs provide a favorable environment for neural regeneration and suggests that this technique has therapeutic potential for promoting RLN recovery. Therefore, when an RLN is invaded by a tumor or injured during surgery, intraoperative reconstruction of the RLN is attempted3. Surgical options include (1) RLN primary repair  and (2) nerve transplantation, such as free nerve grafting or end-to-side anastomosis5. However, primary repair is only applicable in cases with RLN defect lengths of ≤5 mm and in both stumps without tension. Furthermore, in the case of nerve transplantation, donor site morbidities are inevitable6. Furthermore, misdirected regeneration among nerve fibers is possible, and the vocalis muscles may gradually atrophy during neural regeneration7. Thus, more effective means are required for functional recovery of the vocal cords.During thyroid and thoracic surgery, recurrent laryngeal nerve (RLN) injury can result in vocal cord paralysis (VCP)8. Several studies have addressed the optimal environments for peripheral nerve regeneration13. The most commonly studied methods involve stem cells, growth factors (GFs) containing or eluting materials that stimulate regenerating axons, and implanting nerve guidance conduits (NGCs) and scaffolds at the site of injury15.Regeneration after peripheral nervous system (PNS) injury has better outcomes than that after central nervous system (CNS) injury. The mammalian PNS is capable of axonal outgrowth, which means that substantial functional recovery can occur, while CNS injury results in glial scar formation that inhibits neural growth16. The selective permeability of this membrane prevents the infiltration of inflammatory cells and myofibroblasts, but permits the diffusion of nutrients and oxygen, which are prerequisites for effective nerve revitalization through an NGC17. The efficacy of this system was previously demonstrated in an RLN defect model2 as well as in a rat sciatic nerve defect model16. Recently, a tissue-engineered conduit in combination with neurotrophic factors, GFs, or stem cell release has been used to promote axonal sprouting to increase the regenerative effects16. On the other hand, platelet-rich plasma (PRP) has been reported to have healing effects on tendons, ligaments, muscle, bone, and on the regeneration of peripheral nerves, which have been ascribed to the various GFs it contains19. Although the effects of these GFs on nerve regeneration have been studied20, little is known about the biological effects of PRP on Schwann cells (SCs)21.In a previous study, NGCs manufactured with an asymmetric polycaprolactone (PCL)/Pluronic F127 porous membrane containing pores of different sizes on the inner (nano-sized) and outer (micro-sized) surfaces were used as implantable biomaterials for the regeneration of rat sciatic nerveThe aim of this study was to evaluate the ability of PRP-loaded PCL/Pluronic F127 membrane NGCs to promote peripheral nerve regeneration  and biomechanical characteristics. First, the biochemical moiety of the PCL membrane was characterized by Fourier transform infrared (FT-IR) vacuum spectrometry. The peaks at 2943 and 2866 cm−1 indicate asymmetric −CH2 stretching, while the peaks at 1722 cm−1 indicated C=O stretching. The peak at 1294 cm−1 was related to C–O and C–C stretching. The C–O–C peaks were located at 1240, 1161, and 1100 cm−1 . Analysis of GF concentrations in activated PRP gel samples revealed that TGF-β1 levels were significantly higher than those in the resting state .The validation of PRP gel activation was performed after adding collagen and platelet activation marker, CD62P expression was upregulated in activated PRP as determined by flow cytometry in the collagen-containing gel compared to the collagen-negative sample . However, on day 2, SCs treated with 5% PRP also showed greater cell proliferation than the control (P = 0.04). On day 3, SCs with 5% PRP did not demonstrate a significant difference compared to the control (P > 0.05), but SCs cultured with 10% PRP showed higher proliferation than those cultured with 0% PRP (P = 0.0001). We also compared the effect of different concentrations of PRP on cell migration using a transwell assay. Human SCs treated with PRP at 5% and 10% demonstrated significantly faster migration rates than those without PRP at 8 h in a dose-dependent manner   . At baseline, cells had a typical spindle shape alignment and a bipolar or occasionally multipolar morphology Fig. . The prol P = 0.0. On day l P = 0.0. On day P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. As shown in the schematic illustration in Fig. P = 0.018)  Fig. .Fig. 6EnP = 0.033) at 12 weeks postoperatively  and for ruling out the secretion of NGF from rat PRP. The expression of NGF in Schwann cells was increased only in 5% PRP compared to 0% PRP, whereas only secreted NGF levels from Schwann cells were dose-dependently increased in 5% and 10% PRP compared to non-treated controls. This suggests that PRP might stimulate the NGF secretion by stimulating SCs, and the increased dose of PRP can promote SC proliferation.SCs have been shown to play crucial roles in peripheral nerve regeneration. Following peripheral nerve injury, SCs proliferate, form a Büngner belt, and together with macrophages, devour the debris of denatured axons and myelin. At the same time, SCs express and secrete neurotrophic factors including NGF, neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), and brain-derived neurotrophic factor (BDNF), which play neuron-protective and axon-inductive roles34. The significant effect of PRP on SC migration observed in the present study suggests that neurotrophic factors such as NT-3 and NGF in PRP play essential roles in the promotion of SC migration.After nerve injury, the migration of SCs is the main mechanism that supports nerve regeneration38. MAG is a ligand that binds to an axonal receptor, and this binding activates cyclin-dependent kinase 5 (cdk5) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling and increases the expression of phosphorylated neurofilaments, which in turn, increase axonal maintenance and survival37. Our results showed that PRP enhanced the expression of MAG and MBP, which are major proteins of the myelin sheath38. Yao et al. demonstrated that insulin-like growth factor I (IGF-I) treatment upregulates the gene expression of MBP in an experimental model of encephalomyelitis39 and that in oligodendrocytes, IGF-I affects myelin protein synthesis and myelin regeneration41. IGF-I acts as a neurotrophic factor that promotes peripheral nerve growth and inhibits neuronal and glial apoptosis43. We found that MBP, which was induced by PRP, could protect neurons from apoptosis through the IGF-I in PRP44 and work in a synergetic fashion with all involved other growth factors in PRP, such as platelet-derived growth factor, vascular endothelial growth factor, and epithelial growth factor for nerve regeneration.MAG is a molecule expressed by myelinating cells at the myelin/axon interface which helps to establish the normal formation and maintenance of myelinated axons41. The present study suggests that PRP promotes axonal growth by stimulating SCs.As shown in Fig. 2. In the present study, neural regeneration was greater at 16 weeks than at 8 weeks post-implantation in the PRP group, and axial TEM images revealed the presence of dense axon bundles, suggesting that nerves grew well in the GF-abundant environment provided by PRP. The significance of the role played by GFs within PRP was also highlighted in a rat brain-spinal cord co-culture system, in which PRP supernatant increased in axon sizes and numbers27.In a previous study, PCL/F127 NGC facilitated rapid bridging of a 10-mm nerve gap and promoted neural tissue growth at 8 weeks post-implantation, which was more than what was observed in the silicone tube group48 and results in the regrowth and guiding of axons at a rate of about 1 mm per day and in their reconnection with target organs51. SCs exhibit a striking plastic response to nerve damage and are the first entities to be exposed and detect nerve damage. In a context- and time-dependent manner, transdifferentiated SCs perform a variety of cellular repair tasks that range from phagocytosing myelin debris to secreting neurotrophic and neurotropic factors (laminin). Furthermore, proliferating and migrating SCs form cords and Büngner bands in the proximal and distal nerve segments, respectively.A multicellular and pleiotropic molecular response is activated after peripheral nerve injury. This response interplays with, and is mainly modulated by, injured axons, myelin breakdown products, soluble factors, and hypoxia53 using sciatic nerves showed that the NGC improved the misdirection of axons compared to nerve autografting or direct anastomosis. After nerve injury, the neuromuscular junction (NMJ) results in remodeling, which increases the fragment number, while individual muscle fiber cell membranes spontaneously create fibrillation potential. At the same time, the target muscles gradually atrophy, and muscle wet weight and muscle fiber diameter gradually decrease. Ma et al. showed that even if the SC number normalizes and targets muscle atrophy severely, muscle wet weight is reduced to 15%54. In this study, a slightly smaller vocalis muscle ratio at week 16 compared to that at week 12 represents severe muscle atrophy during nerve regeneration.Poor functional recovery after peripheral nerve injury has been attributed to the misdirection of regenerating axons to reinnervate functionally inappropriate muscles. The RLN is a branch of the vagus nerve  that supplies all the intrinsic muscles of the larynx, including the vocalis, posterior cricoarytenoid, lateral cricoarytenoid, and interarytenoid muscles. Even though we could not evaluate all target muscles, vocal cord movement after evoked stimulation at the proximal part was demonstrated. Several studies55. Third, the proliferation of SCs is important in nerve regeneration, but we did not perform experiments with a neural cell model. However, the effects of PRP on model neural cells have been demonstrated in previous studies. Baklaushev et al. showed that a PRP-derived hydrogel dramatically stimulated proliferation and neuronal differentiation of directly reprogrammed human neural precursor cells due to the complex action of the PRP components and creation of a 3D biomimetic environment56. In addition, Li et al. demonstrated that human adipose stem cells (hASCs) treated with PRP displayed higher levels of neuron-specific enolase and membrane-associated protein-2 compared to the control group. These results indicate that PRP is capable of promoting cell proliferation and neurogenic differentiation of hASCs in vitro57. Finally, we did not compare gelatin-filled NGCs (vehicle control) and PRP-filled NGCs. Further studies are required to address these issues.The present study has several limitations that warrant consideration. First, the gap created in the RLNs was 10-mm, but the maximum gap and appropriate timing of implantation after injury were not evaluated. This NGC model might be typically necessary in cancer resection settings and would be less applicable in the setting of nerve transection. Second, we did not compare the efficacy of neural regeneration between the end-to-end epineural suture group and the PRP-loaded NGC group. However, Ikumi et al. compared autologous grafting using an epineural suture and an autologous graft coated with activated PRP and found that local PRP administration increased regenerative axon diameters and axon numbers in the distal portions in PRP increased SC proliferation and that PRP promoted SCs to secrete neurotrophic factors in vitro, and promoted functional recovery after peripheral nerve injury in vivo. Our findings show that PRP-loaded NGCs provide a favorable environment for neural regeneration and suggest that they offer a possible therapeutic strategy for promoting RLN recovery.In summary, the present study demonstrates the therapeutic potential of an NGC system loaded with PRP for nerve regeneration both in vitro and in vivo. In terms of RLN functional regeneration, PRP-loaded NGCs produced better results than non-loaded NGCs. The PRP group exhibited less vocalis muscle atrophy, faster nerve growth, and greater well-organized axon density. It appears that PRP in NGCs improves neural cell proliferation and regeneration in a GF-rich environment. We found that GFg for 20 min. Platelet number was adjusted to 4-4.5–108/mL by mixing PRP and PPP, and the adjusted PRP preparation was used for the following experiments. The adjusted PRP was incubated at 37 °C with continuous stirring at 1200 rpm. PRP was equilibrated at 37 °C for 3 min before the initiation of the experiment. To activate platelets, collagen (10 µg/mL) was added, and the reaction was stopped at 30 min by cooling at 4 °C. The expression of CD62P (platelet activation marker) was measured using a flow cytometer . Total protein concentration was measured by bicinchoninic acid (BCA) protein assay , while transforming growth factor-beta (TGF-β) was quantified using an enzyme-linked immunosorbent assay  following the manufacturer’s instructions.Rat blood was drawn from the heart using a disposable syringe containing 2.2% sodium citrate . PRP was prepared by centrifugation of citrated blood at 200 g for 10 min at room temperature (22 °C), and platelet-poor plasma (PPP) was obtained from the residue by centrifugation at 1500 × 16. Briefly, to make an asymmetrically porous sheet (nano- and micropores on both surfaces), we dissolved the PCL pellets in tetraglycol  at 90 °C; then, we added Pluronic F127 powder  to the PCL solution . PCL/F127 (50 mm × 50 mm × 0.4 mm) was molded and immersed for 1 h at room temperature. The precipitated PCL/F127 sheet was washed and vacuum-dried. The sheet was rolled into a tube using a 1 mm diameter metal mandrel , and the edge of the sheet was fixed using a tissue adhesive . The prepared asymmetrically porous NGCs had an inner diameter of 1 mm and a length of 10 mm. We validated the chemical moiety of the PCL membrane using a Fourier transform-infrared vacuum spectrometer . The mechanical properties of the PCL membranes were measured using a tensile tester . The specimens were prepared in a dog-bone shape (width: 0.3 mm and length: 0.5 mm).PCL/Pluronic F127 sheets were prepared using an immersion precipitation method, while NGCs were produced by rolling, as previously described58. Briefly, PRP was mixed with 2% alginate solution made from alginic acid sodium salt derived from brown algae . The mixture was then dispensed via a syringe needle (26½ gauge) inside the NGC tube blending in 6% CaCl2 (Sigma). The PRP plus alginate mixture was gelled by the diffusion of Ca2+ ions into the polymer mixture. After gelation, the beads were incubated in CaCl2 solution for 20 min to complete the gelation process.The PRP alginate mixture was fabricated through the internal gelation process, as previously described2)-coated culture dishes. The medium was changed every three days until the culture was approximately 90% confluent.Human SCs were purchased from ScienCell Research Laboratories  and cultured in Schwann cell medium  in poly-L-lysine  on culture days 1 and 3. After adding 10 µL of CCK-8 reagent and incubation at 37 °C for 3 h, formazan absorbance was measured at 450 nm using a well plate reader .Primary SCs were seeded in 96 well plates at 1 × 10Cell migration was assessed using a transwell assay  and wound healing assay. For transwell assays, SCs were introduced into top chambers in culture medium and different concentrations  of PRP gel were added to the lower chambers. After 4 and 8 h, non-migrated cells on the upper membrane surfaces were removed using cotton swabs, while migrated SCs on lower surfaces were fixed with methanol and stained with crystal violet solution. Migrated cells were imaged and counted under a microscope . The number of migrated cells was expressed as percentages of migrated non-treated controls.For the wound healing assay, SCs were grown to confluence in SCM containing 0%, 5%, and 10% PRP gel. Confluent layers were then ‘wounded’ using a sterile 100 µL pipette tip, and 4 and 8 h later, migration rates into wounded areas were measured using an inverted microscope . Migration areas are expressed as percentages of those of PRP-untreated controls.Western blotting was performed to assess the changes in protein expression in human SCs treated with PRP at 0%, 5%, and 10% concentrations. The primary antibodies used were as follows: myelin-associated glycoprotein , myelin basic protein , neurotrophin-3 , nerve growth factor , and extracellular signal-regulated kinase . GAPDH  was used as an internal control. All blots derive from the same experiment and were processed in parallel. Quantitative analysis was performed using ImageJ software . To rule out the expression of NGF in rat PRP, NGF concentrations in SC-media were determined by ELISA  according to the manufacturer’s instructions.n = 10) or a PRP-loaded NGC tube group (n = 12). The right RLNs of rabbits were preserved as normal controls, but the left RLNs were resected and interposed with an NGC  and animal care and experiments were performed in accordance with established institutional animal ethics committee regulations. Twenty-two female New Zealand white rabbits about 2 kg at 12 weeks of age were randomly assigned to a non-loaded NGC group . Then, we stimulated a 2-mm site proximal to the resected region using a nerve stimulator  with an electric current of 1.5 mA. After nerve stimulation, we checked vocal cord movements, captured images in adducted and abducted positions, and calculated mean vocal cord gap ratios  in the non-loaded NGC and PRP-loaded NGC groups. Restoration of vocal cord movement was evaluated using a binary scale (yes/no). We also calculated the relative gap ratio (left gap/right gap × 100), and the gap was measured with the triangular areas in the fully abducted and adducted positions using Image J .After the endoscopic vocal cord evaluation, the animals were sacrificed. NGCs, including RLNs and nerve stumps, were harvested to evaluate nerve growth, and larynges were resected to assess the vocalis muscle status. NGC tubes including RLNs and larynges were fixed in 4% formaldehyde, immediately embedded in paraffin, and sectioned . Longitudinal sections of NGCs were stained with hematoxylin and eosin (H&E) and toluidine blue, while vocal process level of the vocalis muscles were stained with H&E. A single-blinded observer measured the cross-sectional areas of the vocalis muscles using ImageJ software by tracing the outlines of the microscopic images. Areas of the left side denervated vocalis muscles were expressed as percentages of those of the normal right side muscles.To identify specific neural markers in regenerated tissue inside NGCs, we performed immunohistochemical analyses using AChE , neurofilament , and anti-S100 protein .Regenerated nerve structures were examined by transmission electron microscopy  to investigate regenerated nerve structures, especially the transverse section of the NGC at 8, 12, and 16 weeks postoperatively. The number of axons was counted, and the overall structures of myelinated fiber bundles were noted.p values < 0.05.The Kruskal Wallis and Mann–Whitney tests were used to determine the significance of the effects of different PRP concentrations on proliferation, migration, wound healing, and the expression of target proteins. The Mann–Whitney test was used to determine the difference in vocal cord movements, axial section areas of the vocalis muscles, and number of nerve endings in the control and PRP groups. The analysis was performed with GraphPad Prism 5.0 , and statistical significance was accepted for Further information on research design is available in the Supplemental materialsVideo S1Video S2REPORTING SUMMARY


Body fat (%) was significantly decreased in the two protein groups. Fat-free mass did not significantly change among the three groups. Serum metabolomics showed that the tricarboxylic acid cycle pathway was upregulated in the WPH group. No significant changes in microbiota were observed among the groups. In conclusion, WP or WPH supplementation combined with an energy-restricted diet benefits older women during weight loss. WPH was more effective, possibly due to increased energy metabolism.An energy-restricted weight-loss approach has limitations when it used in the elderly, especially because of muscle loss. We aimed to assess the effects of whey protein (WP) or WP hydrolysate (WPH) combined with an energy-restricted diet (ERD) on weight reduction and muscle preservation in older women with overweight and obesity. A total of 60 women were randomized to the control (ERD), WP (ERD + 20 g/d WP) or WPH (ERD + 20 g/d WPH) group, using a 1:1:1 allocation ratio. After an 8-week intervention, body composition, gut microbiota, and serum metabolomics changes were compared among the three groups. The reductions in body weight (−1.11 ± 1.11 vs. −2.34 ± 1.35,  Obesity has become a major global public health issue. Overweight and obesity have increased rapidly in the past four decades in China, and the latest national prevalence rate was 34.3% for overweight and 16.4% for obesity in adults [Weight loss is important for older adults with obesity, but a conventionally adopted low-calorie diet is likely to exacerbate the age-related loss of skeletal muscle (sarcopenia). Unfortunately, the mortality risks of muscle loss may outweigh the potential benefits of weight loss in the elderly . ResistaIntervention studies in Western countries have shown that high protein intake attenuates muscle mass loss in the natural progress of aging  and duriEmerging evidence showed that gut microbiota is an important factor linked to obesity by altering host energy balance . Diet ca2) were recruited from local communities via advertisement and word-of-mouth from September 2019 to October 2020. Exclusion criteria included: (1) those with digestive diseases, such as gastrointestinal ulcers, inflammatory bowel disease, pancreatitis, severe vomiting, diarrhea, intestinal obstruction, or other conditions that affect gastrointestinal digestion and absorption; (2) those with severe cardiovascular disease, such as heart failure, uncontrolled blood pressure , myocardial infarction, and severe arrhythmias; (3) those with severe pulmonary disease, major liver dysfunction, cognitive impairment, or uncontrolled blood glucose that could adversely influence the results; (4) those with renal insufficiency ; (5) those who were unwilling to eat the prescribed diets, disagreed with maintaining their activity level throughout the study, recently on weight loss, or using supplements such as protein powder, fish oil, antioxidants, and anti-inflammatory products.Older women aged from fifty to eighty years with overweight and obesity  and WPH  were packaged in 10 g per sachets . All theAt baseline, energy-restricted diet and intervention conduction, participants were asked to record their food consumption (two weekdays and one weekend day). Macronutrient content of the diet was assessed using Golden Key Nutrition Expert System . Daily physical activity was evaluated using the physical activity scale for the elderly (PASE) . ProfessThe trial was approved by the Ethics Committee of Soochow Universityand was registered at the Chinese Clinical Trial Registry with the registration number ChiCTR-IPR-2200057152. Written informed consent was obtained from each participant.Participants were required to wear light clothing and remove all metal objects from their bodies while undergoing this test. Body composition, including body fat, visceral fat, fat-free mass, and muscle mass, was measured by inBody S10 .The participants were asked to sit quietly for at least 15 min, and blood pressure (BP) was measured three times at 5 min intervals using an automatic BP measuring device . Additional measurements were taken when BP values varied by ≥10 mmHg.For the biochemical analysis, venous blood samples were collected after overnight fasting of at least 10 h. The serum was separated through centrifugation and stored at −80 °C. Fasting blood glucose (FBG), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and uric acid (UA) were measured using a Hitachi Automatic Biochemical Analyzer 7180  was measured using a fully automatic electrochemiluminescence analyzer Roche Cobas E602 . Insulin sensitivity was assessed by homeostasis model assessment–insulin resistance (HOMA-IR), which was calculated as follows: HOMA-IR = FBG (mmol/L) × FINS (μU/mL)/22.5.Circulating markers of inflammation include C-reactive protein concentration (CRP), tumor necrosis factor α (TNF-α), and free fatty acids (FFA). CRP was determined using an automatic electrochemiluminescence analyzer Roche Cobas C702 . TNF-α and FFA were determined using the multifunctional enzyme marker Tecan sunrise .v/v) and shaken for 3 min to precipitate protein. After centrifugation at 18,000 rpm at 4 °C for 10 min, 120 μL supernatant was absorbed and transferred to the vial for sample injection. Another 20 μL of each sample was taken, and the mixed samples were used as QC samples after processing according to the above method. Before the analysis, three consecutive samples were injected to ensure the good stability of the instrument. QC samples were added between every 10 samples during the analysis for data correction.Metabolomics were determined using liquid chromatography–mass spectrometry . First, 50 μL serum was added to 200 μL mixed solvent CE was 30 with a resolution of 17,500.LC-MS analysis conditions: Chromatographic conditions: Waters HSS T3 column . The mobile phase consisted of 0.1% formic acid water (A)-acetonitrile (B), and the volume flow rate was 0.3 mL/min. The column temperature was 40 °C. The injection volume was 2 μL. Ms conditions: ionization mode was electrospray (ESI) positive ion mode and negative ion mode, the ion source voltage was 3500 V (positive ion) and −2500 V (negative ion), capillary temp was 325 °C, and aux gas heater temp was 300 °C. The scanning range of primary mass spectrometry parent ion was 100–1500 Stool samples were collected at baseline and end of the intervention. After normal defecation, a small amount of fresh feces was collected from a toilet paper with a swab. The cotton swabs stained with feces were immersed in the liquid of the sampling tube and stirred to elute the feces into the preservation liquid of the sampling tube. Stool collection was completed by the subjects according to the standard collection method. The researcher stored the feces samples in the −80 °C refrigerator. Afterward, DNA was extracted using a sodium dodecyl sulfonate (SDS) lysate freezing–thawing method. Total bacterial genomic DNA was extracted using the GHFDE100 DNA isolation kit  according to the manufacturers’ instructions. The quantity and quality of the extracted DNA were measured using a NanoDrop ND-1000 spectrophotometer  and agarose gel electrophoresis, respectively. The above process was completed by the Hangzhou Guhe Information Technology Co., Ltd. (Hangzhou China)The 16S rRNA sequencing was performed by Hangzhou Guhe Information Technology Co., Ltd. The polymerase chain reaction (PCR) was performed as described . The PCRhttp://huttenhower.sph.harvard.edu/galaxy/, accessed on 20 March 2022.).The sample size of the trial was estimated using PASS software based on a previous study from our team. Subjects who completed the study were included in the data analysis. The statistical analysis software used in the research was open source R4.02 software and SPSS26.0 . The graphs were drawn by GraphPad prism 8. Data were expressed as mean ± SD  or median (interquartile range) . Comparison among the three groups was performed by one-way ANOVA or Kruskal–Wallis H test. Further, comparisons between groups were performed using LSD or the Mann–Whitney U test. Paired t-test or Wilcoxon signed-rank test were used to compare differences before and after the intervention. One-way ANOVA and LSD were used to compare the α diversity among the three groups, and paired t-test was used for comparison before and after the intervention. Kruskal–Wallis H test and the Mann–Whitney U test were used to compare the gut microbiota composition among the three groups, and Wilcoxon signed-rank test was used for comparison before and after the intervention. LDA Effect Size (LEfSe) analysis (LDA > 2) was performed on the website  website for metabolic pathway analysis, and metabolic pathways with effect value greater than 0.08 were considered as potential target pathways.Analysis of metabolomics: Using compound discovery software to preprocess the original data, such as peak identification, alignment, noise filtering, peak area normalization, and compound identification, a data matrix containing metabolite retention time, mass charge ratio, and intensity was obtained. The results were imported into SIMCA-P 13.0 software for principal component analysis (PCA) and orthogonal partial least squares–discriminant analysis (OPLS-DA). Variables that meet the projected value of variable importance (VIP) > 1.0 and FDR < 0.05 were considered different variables. The different variables were entered into the MetaboAnalyst 5.0  completed the study.2 (SD = 1.61), respectively. We collected the dietary intake and activity levels of the participants via dietary surveys and physical activity scales. The results showed that the average energy intake was 1458.17 kcal (SD = 301.23), with 13.3% from protein, 61.0% from carbohydrate, and 25.7% from fat. The average PASE was 112.98 . After randomization, no significant differences were noted in baseline body weight among the three groups. No significant differences were noted in body weight among those who completed or did not complete the study in the three groups. The baseline characteristics of the subjects who completed the study are shown in p < 0.01) and WPH  vs. 1003.00 , p < 0.01) groups than in the control group because of the additional protein supplements given in WP and WPH groups  vs. 1003.00 , H groups .p < 0.05), BMI , and body fat . The percentage of body fat and the visceral fat area were significantly decreased in the WP and the WPH groups after the 8-week intervention. The WPH group had lower decreases in the percentage of body fat and visceral fat areacompared with the WP group, but the differences were not statistically significant. Fat-free mass did not differ statistically after the intervention in the three groups, although there was an increasing tendency for fat-free mass in the WPH group.   . Among t < 0.05) . The cooIn the α diversity analysis, no significant change was observed after intervention among the three groups based on Chao1, Ace, and Shannon . No signLoss of skeletal muscle (sarcopenia) progresses with age and is a tough issue in the elderly with obesity. Caloric reduction combined with appropriate protein supplementation may prevent muscle loss. Therefore, we compared the effect of WP and WPH supplementation in an energy-restricted diet on body weight and muscle mass in older women with overweight and obesity. We found that an eight-week energy-restricted diet significantly decreased body weight and fat mass, with more noticeable results in the WPH group. Fat-free mass was preserved to a great extent in the WP and WPH groups (vs. the control group), but the change was not significant. Similar to our trial, Coker et al.  conducteIn the present study, WPH, instead of WP, supplementation had a BP-lowering effect. On the one hand, the weight loss in the WPH group was greater than in the WP group, and the greater weight loss was accompanied by an improvement in blood pressure. On the other hand, It is reported that milk-derived peptides have a hypotensive effect via inhibition of the angiotensin-converting enzyme (ACE) . For the metabolism of glucose and lipids, WP supplementation significantly decreased serum TG. Rakvaag et al.  reportedThe tricarboxylic acid (TCA) cycle plays an important role in metabolism. The fluency of the TCA cycle is very important for maintaining normal cellular function, especially energy output . A previThe gut microbiome has been viewed as a predictor of health, and the environment and diet can influence the composition or function of the gut microbiome ,31,32. AAs far as we know, this study is the first trial conducted on the Chinese elder population to observe the whey protein effect in a weight-loss intervention. The protein intake of the elderly in China is lower than that in western countries. In our study, an energy-restricted diet was recommended according to the ideal body weight of individuals, which was more accurate than a consistent calorie recommendation. However, our study has some limitations: (1) the bitter taste of hydrolyzed protein makes double-blind design impossible; (2) although personalized dietary guidance was instructed by the dietitian, standard meals were not provided for participants; and (3) high interindividual variations in the gut microbiota may mask minor changes in distinct taxonomic diversity.In conclusion, an energy-restricted diet reduced weight and fat mass in older women with overweight and obesity. WP and WPH supplementation further improved body composition. WPH was more effective in this improvement, and its mechanism is possibly associated with increased energy metabolism.