Persistent cold temperatures, a paucity of nutrients, freeze-thaw cycles, and the strongly seasonal light regime make Antarctica one of Earth’s least hospitable surface environments for complex life. Cyanobacteria, however, are well-adapted to such conditions and are often the dominant primary producers in Antarctic inland water environments. In particular, the network of meltwater ponds on the ‘dirty ice’ of the McMurdo Ice Shelf is an ecosystem with extensive cyanobacteria-dominated microbial mat accumulations. This study investigated intact polar lipids (IPLs), heterocyte glycolipids (HGs), and bacteriohopanepolyols (BHPs) in combination with 16S and 18S rRNA gene diversity in microbial mats of twelve ponds in this unique polar ecosystem. To constrain the effects of nutrient availability, temperature and freeze-thaw cycles on the lipid membrane composition, lipids were compared to stromatolite-forming cyanobacterial mats from ice-covered lakes in the McMurdo Dry Valleys as well as from (sub)tropical regions and hot springs. The 16S rRNA gene compositions of the McMurdo Ice Shelf mats confirm the dominance of Cyanobacteria and Proteobacteria while the 18S rRNA gene composition indicates the presence of Ochrophyta, Chlorophyta, Ciliophora, and other microfauna. IPL analyses revealed a predominantly bacterial community in the meltwater ponds, with archaeal lipids being barely detectable. IPLs are dominated by glycolipids and phospholipids, followed by aminolipids. The high abundance of sugar-bound lipids accords with a predominance of cyanobacterial primary producers. The phosphate-limited samples from the (sub)tropical, hot spring, and Lake Vanda sites revealed a higher abundance of aminolipids compared to those of the nitrogen-limited meltwater ponds, affirming the direct affects that N and P availability have on IPL compositions. The high abundance of polyunsaturated IPLs in the Antarctic microbial mats suggests that these lipids provide an important mechanism to maintain membrane fluidity in cold environments. High abundances of HG keto-ols and HG keto-diols, produced by heterocytous cyanobacteria, further support these findings and reveal a unique distribution compared to those from warmer climates. Antarctica is replete with environments where complex life is excluded. Sub-zero temperatures, the scarcity of liquid water, nutrient limitation , and str2  when Earth experienced two long-lasting glaciations interspersed with periodic ‘hothouse’ conditions as articulated in the ‘Snowball Earth’ hypothesis . During Lipid biomarkers, such as intact polar lipids (IPLs), heterocyte glycolipids (HGs), and bacteriohopanepolyols (BHPs), have been extensively applied to investigate the chemotaxonomic composition of metabolically active microbial communities. IPLs comprising a polar head group bound to a glycerol backbone and linked to a hydrophobic hydrocarbon tail, encode valuable chemotaxonomic information . HGs comThe high biomass and low anthropogenic interference in these meltwater ponds, coupled with climatic conditions unique to the Antarctic, constitute an ideal natural laboratory to study homeoviscous adaption strategies of the microbial cell membranes. However, to date, only one study has investigated the lipid distribution of actively growing microbial mats in three McMurdo Ice Shelf meltwater ponds , while aNostoc spheres’ and a ‘sheet,’ which lay on top of the microbial mats from four ponds , where they were lyophilized, shipped to Cambridge (United States), and then maintained at −20°C until lipid extraction. The physicochemical conditions of the meltwater ponds , Lake Fryxell  and Lake Vanda tropical and hot spring environments, additional samples were investigated from Highborne Cay , Hamelin Pool in Shark Bay , and Yellowstone National Park . The mats from Highborne Cay were collected in 2010 from a tidal flat and included some encrusted algae. The Hamelin Pool samples were taken from a stromatolite-forming cyanobacterial mat  in June 2011. The Yellowstone sample was collected in June 2012. All samples were stored in sterile Whirl-Paks immediately after sample collection at −20°C and freeze-dried before extraction.–1), 4 μL of 5X GoTaq reaction buffer (Promega), 2 μL of MgCl2, 0.16 μL of 200 μM dNTP, 0.2 μL GoTaq G2 DNA polymerase (Promega) and 1 μL of each primer (10 μM). DNA was amplified using the following thermocycler program: 94°C for 3 min, followed by 30 cycles of 94°C for 45 s, 50°C (16S rRNA gene amplification) or 56°C (18S rRNA gene amplification) for 60 s, 72°C for 90 s and a final extension at 72°C for 10 min. In the following, PCR products were purified using AxyPrep Mag PCR Clean-up Kits (Axygen), and triplicate products were combined. The concentration of the purified PCR products was measured in duplicate with a Qubit 2.0 Fluorometer (Thermo Fisher Scientific), and 16S and 18S rRNA gene amplicons were pooled separately at equimolar concentrations for high throughput sequencing. The samples were sequenced (2 × 250 cycles) on an Illumina Miseq platform at the Natural History Museum sequencing facility (United Kingdom). The sequencing data has been deposited at SRA (BioProject ID: PRJNA 758785).DNA was extracted using the PowerBiofilm DNA isolation Kit (MO BIO Laboratories). Archaeal and bacterial 16S small ribosomal subunit rRNA (260 bp) was sequenced by the primers utilizing 515 F (5′-TGGCCAGCMGCCGCGGTAA -3′) and 806R (5′- GGACTACNVGGGTWTCTAAT -3′) , 2012. E16S and 18S rRNA gene sequences were analyzed using the QIIME 2 bioinformatics platform, version 2019.4 . The demNostoc spheres’ were homogenized. IPLs, HGs, and BHPs were extracted four times following a modified Bligh and Dyer protocol , while in steps three and four, trichloroacetic acid  was added to the aqueous phase. The sediments were extracted two additional times with DCM and MeOH  to enhance the extraction efficiency of less polar compounds. The samples were sonicated for 20 min in an ultrasonic bath during each extraction step. The sonicated sediment was then centrifuged at 175 relative centrifugal force (rcf) for 10 min, and the supernatants were combined in a separatory funnel. Deionized Milli-Q water was added to the funnel to allow phase separation. The aqueous solution was extracted three times with DCM. Finally, the organic phase was washed three times with deionized Milli-Q water, and the total lipid extract (TLE) was gently dried under a stream of N2 and stored at −20°C until further processing.Before lipid extraction, lyophilized mats and ‘protocol  with met–1 and 40°C throughout the analysis. The gradient started with an isocratic flow of 5% B for 4 min. The proportion of eluent B gradually increased to 25% at 22.5 min and 40% at 26.5 min. Eluent B was held at 40% for 1 min, and then the column was re-equilibrated with 5% B for 8 min. The mass spectrometer was operated in positive ionization mode, scanning 200 – 2,000 daltons. IPLs and HGs were identified based on their exact mass, retention time, and characteristic fragmentation patterns  with an electrospray ionization source coupled to a 1200 series high-performance liquid chromatograph . IPLs and HGs were separated using a Waters Acquity amide column  used in hydrophilic interaction mode, as described previously . Eluent patterns . IPL and–1. Eluent A contained a mixture of MeOH and HPLC-grade water  and eluent B consisted of isopropanol (100%). The gradient started with 0% B for 2 min, increased to 20% at 20 min, stayed constant for 10 min, and further increased from 20% B (30 min) to 30% B at 40 min. Thereafter, the column was reconditioned for 15 min with 0% B. BHPs were identified based on their accurate mass, retention time, and characteristic fragments (https://doi.org/10.48667/r1zs-v785).An aliquot of each TLE was acetylated with 40 μL of a mixture of pyridine and acetic anhydride  at 70°C for 1 h and left overnight at room temperature prior to the analysis of BHPs. The analysis was performed using the same HPLC-MS system and methodology as described by ragments , 2008. Bragments . Due to Variation of the microbial community between the different ponds were analyzed by a principal coordinates analysis (PCoA). PCoA analysis for Bacteria, Archaea, Eukaryotes and IPLs was calculated by Bray-Curtis dissimilarities matrices using the “vegan” package in R v. 4.0.4  and projX:Y in the DBI refers to the peak area of the compound with x carbon atoms and y unsaturation. The peak area of the compound was multiplied by the number of unsaturation and divided by the total peak area of all compounds (Eq. 1). The ACL was calculated by multiplying the number of x C-atoms in the side chain with the peak area of the compound, divided by the total summed peak area of all compounds . To account for varying chain lengths of the different IPL species , the number of unsaturations was normalized according to their average chain length .To evaluate the membrane properties, the double bond index (DBI) and the average chain length (ACL) of IPLs were calculated as described by Nostoc spheres.’ Prior lipid analysis in these environments screened for hydrocarbons, sterols, and fatty acids  and Bacteroidetes 1–30%) based on 16S rRNA gene sequences . Proteobphagales , 3. A pr0% based phagales . Minor gphagales . This agphagales . While sphagales . FurtherArchaeal 16S rRNA gene sequences were only detected in Pond Seventy and Salt Pond and represented less than 0.01% of 16S rRNA gene sequences . This suNostoc sphere’ samples revealed a high relative abundance of rotifers compared to the other samples, suggesting that these microscopic animals are grazing on the cyanobacteria. In addition, these samples had a lower proportion of photosynthetic eukaryotes, which were primarily of the families Chlorophyceae and Bacillariophyceae. The other microbial mats from the meltwater ponds revealed a more heterogeneous distribution of eukaryotes, additionally featuring a higher proportion of protists such as the Amoebozoa (Discosea and Tubulinea) and ciliates. PCoA of the 18S rRNA gene sequences revealed no distinct eukaryal assemblages in the microbial mats from the meltwater ponds, consistent with previous observations that eukarya on the McMurdo Ice Shelf have a broad tolerance to the local environmental conditions , while other IPLs were only present in trace amounts  or as sheets (∼55% G-DAGs), could affect the lipid composition in Nostoc communities. Future research is needed to investigate whether different growth patterns might affect the lipid composition in these microbes. Moreover, the PCoA showed no pronounced differences in the IPL distribution between fresh and brackish meltwater. This implies that the low to medium salt concentrations have little effect on the lipid composition of microbes in Antarctic meltwater ponds.Principal coordinates analysis of the IPLs showed separate clusters of the ‘ial mats . The IPL amounts . The IPLcultures . SurprisThe microbial mat from the Skua Pond showed an IPL distribution distinct from the communities from the other meltwater ponds . Here, p26 diols and keto-ols were most abundant and constituted between 70 and 100% of all HGs. Particularly, mats dominated by Nostoc spp. (‘Nostoc spheres’ and Fogghorne Pond) and Nodularia spp.  were only detected as minor compounds <10%,  and, notf Nostoc . It was response . For insresponse . The higresponse , the taxscovered .N/P ratios, commonly between 1 and 3, have been observed in the meltwater ponds , indicating that cyanobacteria likely play an important role in these settings. This agrees with previous DNA based studies showing that cyanobacteria are among the most abundant bacteria in Highborne Cay, Shark Bay, and the investigated Antarctic lakes . Howeverer ponds , and Howreported . This sureported . A replareported . Genes freported .Besides the IPL head group composition, the properties of the core fatty acids were investigated. This analysis revealed that the acyl chains in the (sub)tropical microbial mats were more saturated than in samples from Antarctica . These oin situ temperatures , whereas a higher median PAR of ∼45% was determined for 39 different meltwater ponds on the McMurdo Ice Shelf during austral summer . Therefontensity . Furtherntensity . Howeverntensity .26, a HG based proxy to reconstruct surface water temperatures (SWT) in lacustrine environments  and lakes  compared to the (sub)tropical mats . HG distribution patterns and the HDI26 thus express a significant climate-driven component, which may compensate for varying rates of oxygen diffusion into the heterocyte with temperature variation and provide cyanobacteria with an anaerobic microenvironment to allow for biological N2 fixation  compared to the (sub)tropical and hot spring samples . In generonments , 2021, rfixation .The microbial mats from the McMurdo Ice Shelf showed substantial variations of the BHP distribution pattern in the different meltwater ponds, which complicated the comparison with samples from other environments . In geneS-adenosylmethionine protein that catalyzes the methylation of hopanoids at the C-position, a recent study suggested that 2-Me-BHPs in Lake Vanda are produced by cyanobacteria tropical environments. The microbial mats from Lake Vanda showed a high abundance of 2-Me-BHPs (30 – 35%). Such a high abundance of methylated BHPs was not detected in any other investigated environment. Based on genetic analysis of the radical bacteria . Given tbacteria , were debacteria  was dete2. Samples from the Bratina meltwater ponds originated from waters that have previously been shown to be oversaturated (>625 μM) with respect to O2 tropical and hot spring environments; likely an adaption at low temperatures to maintain the integrity of the gas diffusion barrier and limit the inflow of O2 into the heterocyte.The microbial mat communities in twelve meltwater ponds from the McMurdo Ice Shelf were investigated for their IPL, HG, and BHP contents and compared with 16S and 18S rRNA gene community compositions. IPL distribution patterns dominated by glycolipids are in accordance with the predominance of cyanobacteria in these environments. Our results revealed a high abundance of phospholipids in microbial mats from the meltwater ponds, whereas aminolipids substitute these in P-limited systems. Comparison with microbial mats in perennially ice-covered Antarctic lakes, (sub)tropical, and hot spring environments revealed that the microbes in the meltwater ponds produce highly unsaturated lipids likely to increase the fluidity of the lipid membrane in cold environments and protect the inner cell against high levels of solar irradiance during austral summer. Similarly, HGs produced by NThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/RES, IH, and TWE designed the research. TJM, IH, and RES collected samples. TWE, MJK, ADJ, TB, and TJM performed the research. TWE, ADJ, JLM, TB, and HG analyzed the data. TWE and RES wrote the manuscript with contributions from all co-authors. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Vitis vinifera cvs. Chardonnay, Pinot Noir, Cabernet Sauvignon, and Vitis sp. cv. Koshu, grown in eleven vineyards in Japan. The number of endophytic fungal species was small in the grapevine shoot xylems and could not be analyzed further, whereas a total of 7,019,600 amplicon sequences  and 1305 bacterial operational taxonomic units were obtained by analysis of the V3–V4 region of the bacterial 16S rRNA gene. Gammaproteobacteria was predominant in the shoot xylems at the shoot elongation stage irrespective of the cultivar, whereas Alphaproteobacteria and Oxyphotobacteria were predominant at véraison. Actinobacteria, Bacteroidia, Bacilli, and Clostridia were also detected in the shoot xylems. The endophytic bacterial microbiota in Koshu and Pinot Noir shoot xylems were similar irrespective of the grapevine-growing region. In contrast, the endophytic bacterial microbiota in Chardonnay and Cabernet Sauvignon showed diversity and complexity among grapevine-growing regions. Alpha diversity analysis revealed that Koshu shoot xylems had a higher diversity of endophytic bacterial microbiota than Pinot Noir, Chardonnay, and Cabernet Sauvignon shoot xylems, and that grapevine shoot xylems at the shoot elongation stage had a higher diversity of endophytic bacterial microbiota than those at véraison. Principal coordinate analysis (PCoA) demonstrated that the profiles of the endophytic bacterial microbiota in grapevine shoot xylems at véraison were relatively uniform compared with those at the shoot elongation stage. Multidimensional scaling analysis showed that the plots of all cultivars were generally apart from each other at the shoot elongation stage and then became close to each other at véraison. The plots of all grapevine-growing regions cultivating Koshu were close to each other, whereas those of grapevine-growing regions cultivating Chardonnay and Cabernet Sauvignon were apart from each other. The findings of this study suggest that the endophytic bacterial microbiota in grapevine shoot xylems varied depending on the cultivar and the grapevine-growing region even for the same cultivars, and that the microbiota fluctuated depending on the shoot growth stage.Next-generation sequencing technology may clarify microbiota that are as yet poorly understood in the soil, the rhizosphere, and the phyllosphere of vineyards. To provide new information on the interaction between grapevine and microorganisms, we focused on the endophytic microbiota in grapevine. We performed endophytic microbiome analysis of the shoot xylems of four cultivars,  In Japan, the characteristics of berries of Koshu, which is a Japanese indigenous Vitis sp., vary among the grapevine-growing regions having different climatic conditions5. For example, the average monthly air temperature showed a strong relationship with total soluble solid content, titratable acid content, fresh berry weight, total phenol content, caftaric acid content, and coutaric acid content in Koshu berries. Total integrated solar radiation also showed a positive correlation with soluble solid content and pH and a negative correlation with titratable acid content in Koshu berries. However, multiple complicatedly intertwined geological and meteorological factors have made it difficult to understand the effects of environmental conditions on the quality of grape berries and wine flavor and aroma.Environmental conditions in each grapevine-growing region may affect the quality of grape berries and cause a diversity in wine flavor and aroma even for the same grape cultivars. Soil composition6. Interestingly, environmental factors such as soil composition7, topography8, and climate9 synergistically affect soil microbiota. Soil type10 and pH11 are the key factors determining bacterial microbiota in arable soils. Topography and climate, such as rainfall pattern and temperature, affect soil microbiota in vineyards through their impacts on soil8. Thus, distinctive soil microbiota in each vineyard may result in the diversity of grape berry and wine qualities among grapevine-growing regions. However, the complete composition of soil microbiota cannot be determined using current technologies owing to the enormous number of microbial species and populations in soils12. The relationship between soil microbiota and grape berry and wine qualities requires further examination.Some scientists have been paying attention to soil microbiota in vineyards to understand the diversity of grape berry and wine qualities among vineyards. Soils contain large numbers of fungi, bacteria, and archaea. Most soil microorganisms remain unidentified and may have unknown physiological and ecological attributes in plant–soil–microorganisms interactions13 or fungus14 within a plant. Endophytism is a mutualistic and win–win relationship between a host plant and an endophyte15. Endophytes frequently alter the physiological status of host plants. Some endophytes stimulate root growth by secreting plant hormones within the host plant16. Colonization by the endophytic fungus Neotyphodium lolii in perennial ryegrass affects the phenolic content in the host grass17. Endophytes also confer tolerance to environmental stresses in host plants. Reactive oxygen species produced by endophytic fungi protect host plants from oxidative stress18. Endophytic Actinobacteria isolated from healthy wheat tissues enhances the disease resistance of Arabidopsis plants by activating systemic defense pathways19. Thus, endophytic microbiota are an active component of host plants’ responses to biotic and abiotic stresses. Optimal endophytic microbiota make host plants healthy, whereas plants with imbalanced endophytic populations may be in poor health.An endophyte is an endosymbiotic bacterium20 and cordon, shoot, and sap21 have been receiving much attention. Minor differences in endophytic bacterial microbiota were detected between shoots of Merlot and Chardonnay cultivated in northern Italy22. Because the diversity of endophytic microbiota within the grapevine may be affected by environmental factors23, the health of the grapevine may also be affected by the diversity and composition of endophytic microbiota associated with the grapevine26. We report here the diversity of endophytic bacterial microbiota in the shoot xylems of four cultivars  grown in eleven vineyards in Japan. We demonstrate that the profiles of endophytic bacterial microbiota in grapevine shoot xylems varied depending on the cultivar and the grapevine-growing region even for the same cultivars, and that they fluctuated depending on the shoot growth stage.Recently, endophytic microbiota in grapevine trunkPlant samples were collected from eleven vineyards located in major grapevine-growing regions in Japan, namely, Urausu in Hokkaido Prefecture, Minamisanriku in Miyagi Prefecture, Katsunuma in Yamanashi Prefecture, Kofu in Yamanashi Prefecture, Kai in Yamanashi Prefecture, Komoro in Nagano Prefecture, Ueda in Nagano Prefecture, Izumo in Shimane Prefecture, Shobara in Hiroshima Prefecture, Saijo in Hiroshima Prefecture, and Omishima in Ehime Prefecture Fig. . The lathttps://www.jma.go.jp/jma/menu/menureport.html). Growing degree days  and precipitation from April 1 to October 31, 2020 are summarized in Supplementary Table Weather data of each vineyard were obtained from the website of Japan Meteorological Agency  and véraison (the end of July to the end of August) in 2020, samples of shoots of The surface of the first internode from the bottom of a shoot was sterilized in 0.1% (v/v) sodium hypochlorite for 3 min at room temperature. The bark and the epidermal tissue were peeled off from the shoot using a sterilized knife. Xylem shavings were obtained using a sterilized grater. One gram of xylem shavings was added to a sterilized 100 mL flask containing 40 mL of phosphate buffer solution (pH 7.4). After shaking at 130 rpm for 3 h at 25 °C, the solution was filtered through a sterilized cotton gauze. The filtrate was transferred to a 50 mL centrifuge tube and centrifuged at 3,000 rpm for 10 min at room temperature. The supernatant was collected into a new centrifuge tube and centrifuged at 10,000 rpm for 10 min at room temperature. DNA was extracted from the pellet using DNeasy PowerSoil Kit  and used as the endophytic microbial DNA.TCGTCGGCAGCGTCAGATGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′ ; reverse primer, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′ . First-round PCR conditions were as follows: after incubation at 95 °C for 3 min, PCR amplification was performed for 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s with a final extension step at 72 °C for 5 min. After cleaning the first-round PCR product using an AMPure XP kit , the second-round PCR was performed using 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems) and Nextera XT Index Kit (Illumina). Second-round PCR conditions were as follows: after incubation at 95 °C for 3 min, PCR amplification was performed for 8 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s with a final extension step at 72 °C for 5 min. After cleaning the second-round PCR product using the AMPure XP kit, the quality and quantity of the amplicon libraries were evaluated using Agilent 2200 TapeStation , and KAPA Library Quantification Kit (KAPA Biosystems) and Step-One-Plus real-time PCR system , respectively.rRNA metagenomic analysis of the endophytic microbial DNA was performed by NODAI Genome Research Center . In a preliminary experiment, the rRNA-ITS region of fungal rRNA was rarely detected in the endophytic microbial DNA. Thus, in this study, bacterial 16S rRNA metagenomic analysis was performed using the endophytic microbial DNA. Briefly, the amplicon library in the V3-V4 hypervariable region of the bacterial 16S rRNA gene was constructed in accordance with the 16S Metagenomic Sequencing Library Preparation protocol . First-round PCR was performed using 2X KAPA HiFi HotStart ReadyMix  and Illumina overhang adapters and universal primers. The nucleotide sequences of the adapters and the primers used in this study were as follows: forward primer, 5′-https://www.ddbj.nig.ac.jp/dra/index.html; accession number DRA012630, hold date: August 24, 2023).The amplicon libraries were sequenced by 2 × 300 bp paired-end reading using the MiSeq platform (Illumina). The sequenced read data were submitted to the DDBJ Read Archive , Chao1 index, and Shannon index in accordance with a method published previously28. To further analyze the diversity of endophytic bacterial microbiota in shoot samples, beta diversity analysis with principal coordinate analysis (PCoA) and weighted UniFrac distance, permutational multivariate analysis of variance (PERMANOVA), and cluster analysis with multidimensional scaling (MDS) were also performed in accordance with methods published previously32.Data analysis and statistical analysis were performed using QIIME2 pipeline version 2019.10The collection of the plant material and related experiments complies with relevant institutional, national, and international guidelines. All methods were carried out in accordance with relevant guidelines and regulations.To determine whether the profiles of endophytic bacterial microbiota vary widely between shoot samples collected from the same grapevine plant or between shoot samples collected from different grapevine plants of the same cultivar grown in the same vineyard, a preliminary experiment was performed. Microbiome analysis demonstrated that the profiles of endophytic bacterial microbiota were similar between two shoot samples collected from the same Chardonnay or Koshu grapevine plant Fig. . In addiGDDs from April 1 to October 31, 2020 demonstrated that Minamisanriku and Ueda belonged to Region III on the Winkler Index and that Komoro, Shobara, and Saijo belonged to Region IV on the Winkler Index  and evaluated Fig. . Below aAt the shoot elongation stage, more than 90% of endophytic bacteria in Chardonnay and Pinot Noir shoot xylems belonged to class Gammaproteobacteria. Oxyphotobacteria was also detected in the shoot xylems albeit at a very low proportion . At véraison, the proportion of Oxyphotobacteria increased and reached 75% and 74% in Chardonnay and Pinot Noir shoot xylems, respectively. Overall, the profiles of endophytic bacterial microbiota were very similar between Chardonnay and Pinot Noir cultivated in Urausu at each shoot growth stage.At the shoot elongation stage, Gammaproteobacteria was predominant in Koshu and Cabernet Sauvignon shoot xylems, although Oxyphotobacteria and Bacilli were detected as well. At véraison, the proportion of Oxyphotobacteria increased and reached 48% and 75% in Koshu and Cabernet Sauvignon shoot xylems, respectively. The proportion of Alphaproteobacteria also increased at véraison . Overall, the profiles of endophytic bacterial microbiota were similar between Koshu and Cabernet Sauvignon cultivated in Katsunuma at each shoot growth stage.Shoot samples of Koshu, Chardonnay, Pinot Noir, and Cabernet Sauvignon were collected from Kofu. At the shoot elongation stage, Gammaproteobacteria was predominant (approximately 90%) in Koshu and Pinot Noir shoot xylems, whereas more than 80% of endophytic bacteria in Chardonnay and Cabernet Sauvignon shoot xylems belonged to class Oxyphotobacteria. At véraison, the profiles of endophytic bacterial microbiota were similar among the four cultivars grown in Kofu, and Oxyphotobacteria was predominant.Irrespective of the shoot growth stage, Gammaproteobacteria was predominant in Chardonnay and Cabernet Sauvignon shoot xylems. Although Gammaproteobacteria was also predominant in the Koshu shoot xylems at the shoot elongation stage, the proportions of Oxyphotobacteria and Alphaproteobacteria increased in Koshu shoot xylems at véraison .Irrespective of the cultivar , the profiles of endophytic bacterial microbiota in shoot xylems were very similar at each shoot growth stage, and Oxyphotobacteria was predominant. More than 80% of endophytic bacteria in the shoot xylems at véraison belonged to class Oxyphotobacteria.The profiles of endophytic bacterial microbiota in shoot xylems at the shoot elongation stage were similar among Chardonnay, Pinot Noir, and Cabernet Sauvignon, whereas the profile in Chardonnay shoot xylems at véraison was different from those in Pinot Noir and Cabernet Sauvignon shoot xylems. Gammaproteobacteria (76%) was predominant in Chardonnay shoot xylem at véraison. In Pinot Noir and Cabernet Sauvignon shoot xylems at véraison, more than 70% of endophytic bacteria belonged to class Oxyphotobacteria.Unlike other vineyards, there was no similarity of profiles between cultivars (Chardonnay and Cabernet Sauvignon) and between shoot growth stages. Gammaproteobacteria and Oxyphotobacteria were predominant in Chardonnay shoot xylems at the shoot elongation stage and véraison, respectively. In Cabernet Sauvignon shoot xylems, Gammaproteobacteria  and Oxyphotobacteria  were predominant irrespective of the shoot growth stage.Similarly to Urausu and Katsunuma, Gammaproteobacteria was predominant in Chardonnay and Cabernet Sauvignon shoot xylems at the shoot elongation stage. The proportion of Oxyphotobacteria increased at véraison; more than 70% of endophytic bacteria in Chardonnay and Cabernet Sauvignon shoot xylems at véraison belonged to class Oxyphotobacteria. Overall, the profiles of endophytic bacterial microbiota were similar between Chardonnay and Cabernet Sauvignon cultivated in Shobara at each shoot growth stage.Similarly to Urausu, Katsunuma, and Shobara, Gammaproteobacteria  was predominant at the shoot elongation stage and Oxyphotobacteria , at véraison. Overall, the profiles of endophytic bacterial microbiota were similar among Koshu, Pinot Noir, and Cabernet Sauvignon cultivated in Saijo at each shoot growth stage.The profiles of endophytic bacterial microbiota in the shoot xylems of Koshu, Chardonnay, Pinot Noir, and Cabernet Sauvignon cultivated in different vineyards were evaluated Fig. . In KoshThe profiles of endophytic bacterial microbiota in the shoot xylems, regardless of the cultivar, at each shoot growth stage were evaluated Fig. . The proOTUs, Chao1 index, and Shannon index were used as indexes of alpha diversity of endophytic bacterial microbiota among cultivars, shoot growth stages, and vineyards Fig. . The medThe medians of OTUs and Chao1 index at the shoot elongation stage were comparable to those at véraison. The median of the Shannon index at the shoot elongation stage (3.0) was higher than that at véraison (1.7), indicating that grapevine shoot xylems at the shoot elongation stage had a higher diversity of endophytic bacterial microbiota than those at véraison.The medians of OTUs and Chao1 index were the highest for Ueda , whereas those were the lowest for Komoro . The medians of the Shannon index were lowest and highest for Minamisanriku (1.2) and Ueda (3.9), respectively. These results suggest that a large number of endophytic bacterial species existed in the shoot xylems of grapevine cultivated in Ueda, and that Ueda had the highest diversity of endophytic bacterial microbiota among the vineyards tested.PCoA demonstrated that the plots of Koshu and Pinot Noir were relatively close to each other at the shoot elongation stage and very close to each other at véraison irrespective of the vineyard Fig. , suggestp-values for all combinations of cultivars exceeded 0.05 (Supplementary Table p = 0.001) between the shoot elongation stage and véraison. Although three of fifty-five combinations of vineyards showed significant differences , there was no significant difference between most of the combinations. These results suggest that the variations of endophytic bacterial microbiota in grapevine shoot xylems greatly depended on the shoot growth stage.PERMANOVA demonstrated that the Cluster analysis of endophytic bacterial microbiota in grapevine shoot xylems in various cultivars, shoot growth stages, and vineyards was performed by MDS Figs.  and 7. CNext, MDS and cladistic analysis of each cultivar in the vineyards were performed Fig. . The dis33. Microbiome analyses in the soil, the rhizosphere, and the phyllosphere of vineyards have been performed at the phylum level35. In general, Firmicutes, Acidobacteria, Bacteroidetes, and Actinobacteria are predominant in the soil. Proteobacteria is abundant in the rhizosphere, whereas large numbers of Proteobacteria, Firmicutes, Bacteroidetes, and Acidobacteria live on grapevine leaves. In the present study, Proteobacteria (Alphaproteobacteria and Gammaproteobacteria), Firmicutes (Bacilli and Clostridia), Actinobacteria (Actinobacteria), and Bacteroidetes (Bacteroidia) were also identified as endophytic bacteria in the shoot xylems of grapevine. On the other hand, Cyanobacteria (Oxyphotobacteria), which has not been detected in the soil, the rhizosphere, and the phyllosphere of vineyards, existed in the shoot xylems of grapevine. V. vinifera cv. Pinot Nero grapevine treated with the extract of Cyanobacteria, Arthrospira platensis F&M-C256, isolated from seaweed showed increased berry weight at harvest compared with untreated grapevine36. In our previous work, we found that the application of Bacillus velezensis KOF112 strain, isolated from the shoot xylems of Koshu, to grapevine leaves induced plant defense response and suppressed downy mildew37. Thus, endophytic bacteria in grapevine shoot xylems are promising candidates for biostimulants to confer desirable traits on grapevine. Further exploration of cultivable endophytic bacteria, which prominently affect grape traits, from grapevine shoot xylems is necessary to stabilize grape berry yield and quality, which are affected by environmental conditions.Microbial communities associated with plants play an important role in plant growth, development, and resistance to external stresses38. The mild impact of environmental conditions on shoot morphology suggests stability in the vegetative growth of grapevine. Therefore, the difference in endophytic bacterial microbiota in the shoot xylems tested may be cultivar-dependent and/or soil condition-dependent. We also found that the profiles of endophytic bacterial microbiota in Chardonnay and Cabernet Sauvignon shoot xylems considerably differed among vineyards. A vineyard is a monoculture cropping system, and the lack of plant genotypic diversity across a vineyard may be a bottleneck in soil microbial diversity21. In addition, plant roots act as ‘gatekeepers’ to screen soil bacteria from the rhizosphere and the rhizoplane33. Regarding Chardonnay and Cabernet Sauvignon, further studies on the relationship between endophytic bacterial microbiota in shoot xylems and soil microbiota in each vineyard are required. On the other hand, the profiles of endophytic bacterial microbiota in the Koshu shoot xylems were highly uniform irrespective of the vineyard, suggesting that endophytic bacterial assemblies in Koshu shoot xylems may not be correlated with environmental conditions in each vineyard including soil microbiota. Koshu is a Japanese indigenous Vitis sp. that has been grown for more than a thousand years in Japan39. The accumulation of phenylpropanoids and flavonols in Koshu grape skin at harvest is one of the distinct characteristics of Koshu compared with other grape cultivars40. The contents of p-coumaric acid, caffeic acid, and flavonols in Koshu grape skin are much higher than those in Chardonnay, Sauvignon Blanc, and Merlot grape skin. The environmental restriction in the grapevine-growing region (in only Japan) for Koshu may have led to the establishment of Koshu-specific endophytic bacterial microbiota in the shoot xylems and contributed to the unique profiles of phenylpropanoids and flavonols in Koshu grape berries. It is necessary to determine the relationship between endophytic bacterial microbiota in Koshu shoot xylems and Koshu grape berry compositions in future experiments.The morphology of each shoot at the shoot elongation stage differed among vineyards and cultivars. Shoot morphology is mainly affected by the genotype and less by the grapevine-growing region41. Commonalities were found between the profiles of microbiota on grape berries and those of soil microbiota in vineyards42. Microbiome analyses of grape musts during winemaking demonstrated that the profiles of microbiota in red grape musts differed from those in white grape musts depending on the cultivar, vintage, and climatic conditions41. Thus, monochronic microbiome analysis of grapevine tissues may not make sense because of the complexity of microbiota in grapevine related to viticulture and winemaking. Comprehensive microbiome analyses, including endophytic microbiota in grapevine from vineyards to winemaking, would help us understand the impact of microbial biodiversity in grapevine on the characteristics of grape berries and wines.Analyses of microbiome in grapevine were also performed from the perspective of winemaking. The surface of grape berries has various microorganisms originating from the environmentSupplementary Figures.Supplementary Tables.


Larger size steel slag (>4.75 mm) and higher cement content are recommended due to heavy metal leaching risk, especially in salty humid areas. Steel slag has sound economic benefits due to the relatively low price. Environmental benefits can also be achieved given that the transport CO2eq emission of steel slag is accounted for. With proper control in production process, steel slag is a very promising alternative material to be utilized in cement-stabilized base course in road engineering.Steel slag has been commonly used in road engineering as cementitious material; however, its application in base course is not widely reported. Four contents of steel slag  were blended into different cement -treated aggregates. Mechanical properties, volume stability, economic benefits and environmental influences of steel slag mixtures were investigated for the feasibility of applying steel slag in semi-rigid base course. Abrasion, crushing and elongated particle content were compared against limestone aggregate, showing that steel slag has the potential of replacing natural aggregate in concrete. Steel slag is beneficial for reinforcement of the strength and stiffness. The mixture has the highest strength and stiffness when bended with 50% steel slag at 4% cement content. By treating steel slag with CH Road infrastructure in China has experienced a fast development in the past three decades. The total mileage of motorway is over 5 million kilometers at the end of 2021, including nearly 160 thousand kilometers of high-speed expressway . For sev−1 based on the current refining processes, the steel slag production is around 60~150 million tons yearly. A huge quantity of steel slag needs to be handled properly. Nevertheless, compared to 85~98% of utilization rate in industrialized countries such as USA, Europe and Japan, the utilization in China is only at 29.5% [Steel slag is one of the by-products generated in steel making process. China’s crude steel production reaches 1.03 billion tons in 2021 [at 29.5% . Most ofThe physic properties of steel slag, i.e., hardness, crushing and grinding, makes it adequate for road engineering. If used appropriately, steel slag can partially or fully replace aggregates in mixtures; not only can the shortage of construction resources be alleviated, but also the damage to environment can be reduced. The utilization of steel slag for road construction in the United States is at nearly 50%; in Europe at 43%, and in Japan at 32.4%, whilst in China it is only at 7.6% ,9. StudiPrevious studies on steel slag in road engineering mainly focus on asphalt mixture and surface course, relatively few on applications of incorporating steel slag in base layer. Based on the reality that cement-stabilized base is the main base type, and the massive slag waste in China at this stage and beyond, the utilizing of steel slag in cement-stabilized base was investigated in this study. We designed four levels of replacement: 0%, 30%, 50% and 75% of steel slag, and the optimal moisture contents were determined through compaction test. Performances of samples with different steel slag contents were evaluated. Unconfined compressive strength, bending strength and resilient modulus were tested for mechanical properties. Volume expansion of steel slag and dry shrinkage of steel slag mixture were also checked. Environmental impacts were evaluated in view of heavy metal leaching and carbon footprint. Other means including SEM, X-ray and FAAS were also employed to check the surface morphology, element distribution and chemical composition to comprehend the strength development and environmental impacts of steel slag in cement-stabilized semi-rigid base. 2O3 and SiO2, accounting for about 84% altogether. It is notable that f-CaO and f-MgO experience steady declines with aging time. Metal constitutes of the steel slag were detected with grinded steel slag powder (<0.125 μm) by the Flame Atomic Absorption Spectrophotometer (FAAS). Heavy metal elements of steel slag with 12 months of aging are displayed in The steel slag used in this study was obtained from Shenglong Steel Mill and the limestone was from a quarry, in Fangchenggang, Guangxi, China, as displayed in The particle sizes of steel slag on site mainly lie between 4.75 mm~19 mm, as Cement-treated aggregates with four steel slag contents of 0% (control group), 30%, 50%, 75% and four cement contents of 3%, 4%, 5%, 6% were formulated. The samples of different steel slag contents were compacted using impaction test method as per JTG E51-2009  to deterThe cylindrical specimens with dimension of Φ 150 mm × 150 mm were adopted for unconfined compressive strength (UCS), unconfined compressive resilient modulus (UCRM) and volume expansion tests. Prism specimens with dimension of 100 mm × 100 mm × 400 mm were used for bending strength (BS) and dry shrinkage tests. The samples were controlled at 98% compactness and at least four duplicates were prepared for each test. Samples for UCS and dry shrinkage test were cured in standard condition  for 7 days, while samples for UCRM and BS test were cured for 28 days. The production and curing process of samples are shown in To investigate the influence of steel slag on strength and stiffness, the UTM-100 servo-hydraulic multi-functional material test system was employed to test unconfined compressive strength (UCS), bending strength (BS) and unconfined compressive resilient modulus (UCRM). When the samples were subjected to UCS test, the loading rate was set to 1 mm/min, while in the BS test, the loading rate was set to 50 mm/min, as per JTG E51-2009 . For the3COOH or silica fume were also tested to clarify their inhibition effect on volume expansion. To explore the shrinkage of mixture with different steel slag contents, after curing in standard condition for 7 days, prism samples were examined for initial mass and length and then set in a condition of 20 ± 1 °C and 60 ± 5 % humidity with both ends measured with dial directors to monitor the length change due to dry shrinkage. The mass and length of samples were recorded every day during the first 7 days, every other day from 7th to 28th day, and on the 60th and 90th day. In the end, the specimens were heated to constant weight. Length and mass of the final state were measured. The volume expansion of steel slag and dry shrinkage of cement concrete with steel slag are in The volume stability test consists of two parts: the volume expansion of steel slag and the dry shrinkage of cement-stabilized aggregates containing steel slag. The safety of highway is affected by the possible expansion caused by f-CaO and f-MgO in steel slag. Steel slag with no aging was compacted at OMC, and soaked in water in the mold with permeable platters at each end. Dial directors were set to monitor the volume increase for 10 consecutive days. Samples treated with CHThe heavy metal pollution of steel slag was examined through precipitation of Cr and V. Influence of particle size, cement content and water salinity were evaluated. An amount of 100 g of steel slag with different particle size from less than 0.075 mm to larger than 26.5 mm a was putA case study of cement-stabilized aggregate base course for a typical four-lane highway with a dimension of 1 km long, 20 cm thick and 32 m wide was conducted to investigate economic gains and greenhouse gas (GHG) emissions. In the definition of cement-treated aggregates, mainly Portland cement and gravel constitute the composition. Water is not considered in the subsequent calculation due to its small amount. The consumptions of steel slag, limestone aggregate and cement were determined based on the compaction experiments . Market 2 (CO2eq) emission, life cycle analysis (LCA) models of cement-stabilized aggregate base course were prepared with SimaPro 9 software. The methodology of LCA consists of 4 stages including goal and scope of the project, inventory analysis, impact assessment and interpretation of results. Correspondently, the research methodology followed for this research is represented in 2eq emission for mix preparation is considered the project’s goal and scope. In inventory analysis, binder materials and aggregates are considered from the Ecoinvent database as per European standards. In the LCA assembly stage, for mix preparation, all the binders, minerals and steel slags are evaluated as per the mix compaction as indicated in To further compare the environmental impacts of cement-stabilized aggregate with steel slag in the aspect of equivalent COThe UCS a, BS Fib and UCR3S/C2S contained in steel slag are hydrated to obtain C-S-H gel [In general, when steel slag content increases from 0 to 50%, the strength and stiffness increases. This is because steel slag is a very strong material with rough surface that strengthens the particle extrusion, which helps form a strong aggregate skeleton to increase the resistance to damage. Moreover, the cementitious components of C-S-H gel , which c3A would also react with CaSO4 to form trisulfide calcium aluminate hydrate (C6AS3H32). The internal voids of cement slurry and surface porosity of steel slag are filled with these two hydration products, and the bonding effect of cement particle is improved. Apparently, the cementitious components contained in steel slag would help increase the hydration products, thereby reinforcing the mechanical performance and durability of the mixture.In the event that gypsum is present in cement clinker, CHowever, when the content of steel slag further increases, the high porosity of steel slag increases the voids in the sample. Some of the cement would be adsorbed and wrapped in the surface porosity of steel slag and insulate from further hydration reaction, which is unbeneficial to the strength formation. In this study, when steel slag increases from 50% to 75%, the 7d UCS experienced a decline of 6~14%, and 28 UCRM a decline of 14~18%, indicating that the adverse effects of porosity began to overtake the positive effects of rough surface and gelling composition when replacement content of steel slag is too high. The recommended steel slag replacement is about 50% based on the test results in this study.3COOH or adding silica fume. As can be seen, the volume expansion has a negative relation with CH3COOH concentration or silica fume content. In this study, volume expansion of steel slag treated with 15% CH3COOH or 4.8% silica fume is reduced very close to 0%. However, the expansion rises again when silica fume is increased to 9.6%; the excessive silica fume downgrades the skeleton structure of aggregate and decreases the interlock of aggregates. To assure the volume stability in the long run, it is recommended that the content of silica fume should be strictly controlled, preferably around 4.5%.The volume changes of steel slag are displayed in 3COOH or silica fume. Moreover, adding silica fume to steel slag during production process when it is still in liquid molten state is an effective solution [To ensure long-term performance, there are several recommendations to control the possible expansion of steel slag: Firstly, steel slag should be detected for active substances . Unreliable source of steel slag with high content of f-CaO or f-MgO should not be used. Secondly, steel slag needs to be treated to mitigate the expansion effects, either by aging for a period of time  and (4)).Cumulative moisture loss rates and shrinkage coefficients and represented in Cr and V leaching of different particle sizes are listed in Precipitations of Cr and V with different cement content are shown in The precipitation of heavy metal was also checked in salty water. The salinity of sea water nearby Fangchenggang is about 2%. Steel slag has shown sound performance following similar procedures of pavement engineering; the economic cost and greenhouse gas (GHG) emission are two other aspects to check. The material and transport costs were calculated for 50% and 30% steel slag replacement, as shown in 2eq emission of different steel slag contents were displayed in 2eq emission is 3.06 × 105 kg. Comparatively, the CO2eq emissions of 1 P of “30% steel slag +70% limestone” and “50% steel slag + 50% limestone” base course are 3.18 × 105 kg and 3.37 × 105 kg, respectively. It is found that in the initial assumption of 100 km transport distance for steel slag the total CO2eq emissions of “steel slag + limestone” mixtures actually are higher than the traditional limestone concrete. The corresponding CO2eq emissions for each procedure in the mixture production are summarized in 2eq emission, accounting for nearly 60% of the total emission. This adheres to our ordinary expectation that energy consumption and pollution from cement production is very high. Unlike the overall life cycle, economic performance is usually less sensitive to transport distance [2eq emission from material transport is non-neglectable, and it grows with steel slag replacement. For instance, the CO2eq emission from material (mainly steel slag) transport accounts for as high as 30% of the total emission for the “50% steel slag + 50% limestone” mixture. The environmental impacts of steel slag depend on the weight of steel slag as well as its haul distance. In this sense, we further calculated the critical transport distance. The critical transport distance is 59 km for “50% steel slag + 50% limestone” mixture, and 74 km for “30% steel slag +70% limestone” mixture. That is, environmental benefits can be achieved if the transport distance is below 59 km for “50% steel slag + 50% limestone” mixture, and below 74 km for “30% steel slag + 70% limestone” mixture.The COdistance , CO2eq e(1)Compared to traditional mineral aggregate , steel slag aggregate has equal or superior engineering properties, including density, abrasion resistance and crushing resistance. Due to higher water absorption and larger gravity, the OMC and MDD increase proportionally as more natural aggregates are replaced with steel slag.(2)Mechanical properties including strength and stiffness are significantly improved by steel slag within 50% replacement due to the increased hydration products and interlocked aggregate structure. However, the porosity and water absorption rate have greater negative effect on strength when steel slag replacement further increases.(3)3COOH or addition of silica fume can relieve the volume expansion of steel slag. Nevertheless, excessive silica fume may be detrimental to the aggregate structure and should be kept within a reasonable content: 3–4.5%.Appropriate treatment with CH(4)Smaller particle size shows higher risk of heavy metal precipitation, and the salty water environment in coastal area adds to it. Large particle size (>4.75 mm) and higher cement content are recommended to reduce heavy metal leaching threat.(5)Steel slag mixture has sound economic gains and potential environmental benefits compared to limestone mixture. However, the transport emission of steel slag should be accounted for to determine an environmental haul distance of steel slag.Steel slag is a promising alternative for natural aggregates in pavement base course. In this study, different steel slag content  was added to cement-treated aggregates. Throughout investigations including mechanical properties, volume characterization, economic benefits and environmental impacts, main conclusions are as follows:Further research would be focused on long-term performances of cement-treated aggregate base containing steel slag in real pavement projects in both the theoretic and engineering aspects.


P = 0.018, cut-off value: 1.2), 0.781  and 0.722 , respectively, suggesting that they have significant prognostic value for lung cancer patients. Our data indicate that Beclin 1, EGFR and ALK genes are associated with the prognosis of patients with non-squamous NSCLC. High Beclin 1 expression and negative EGFR and ALK mutations predict a poor prognosis with PFS.Non-small cell lung cancer (NSCLC) is one of the most malignant tumors. The study was carried out to investigate the prognostic value of Beclin 1, EGFR and ALK for this cancer. Patients diagnosed with non-squamous NSCLC and admitted to our hospital from January 2011 to September 2016 were analyzed. Expression of Beclin 1 and mutation of EGFR and ALK were assessed using polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) and analyzed for their relationship with demographic and clinical characteristics of the patients. Multivariate Cox regression models were applied to analyze the risk factors associated with survival and receiver response curves (ROC) were plotted to determine the prognostic value of Beclin 1, EGFR and ALK for patients with non-squamous NSCLC. Compared with adjacent normal tissue, Beclin 1 expression was elevated in the cancer tissue significantly; assessments of EGFR and ALK mutations showed that out of the 480 patients, 233 (48.5%) and 75 (12.6%) patients had EGFR and ALK mutations. Univariate analysis revealed that Beclin 1 level, EGFR and ALK mutations were associated with lymph node metastasis, TNM stage, tumor differentiation and prognosis, but not with gender, age and smoking status. The Kaplan–Meier survival analysis indicated that low Beclin 1 expression and positive EGFR and ALK rearrangements were associated with higher survival rate and longer progress-free survival (PFS). Multivariate Cox regression analysis showed that Beclin 1, EGFR, ALK mutations, tumor differentiation grade, TNM stage and lymph node metastasis were independently associated with PFS. ROC analysis showed that Beclin 1, EGFR and ALK were significant predictors for PFS; the areas under curve (AUC) for Beclin 1, EGFR and ALK were 0.812 ( Lung cancer is a common malignant tumor which seriously endangers people's health and life. The incidence and mortality rate of the cancer rank the first among the cancers in the world . Even ifWith the rapid development of precision cancer medicine, a better understanding of lung cancer-related gene mutations has allowed to detect mutations in genes such as EGFR, ALK, BRAF, HER2, met, ros1 and RET to stratify patients for better treatments, leading to increased survival time of lung cancer patients after targeted treatments –6. In reThe prevalence of early-stage non–small cell lung cancer (NSCLC) is expected to increase with recent implementation of annual screening programs. According to histological typing, 55% of NSCLC belong to lung adenocarcinoma, 34% are squamous cell carcinoma and 11% are others . ClinicaALK rearrangements has become a very important process in treatment decision making for advanced NSCLC ). Means of 2 continuous normally distributed variables were compared by independent samples Student's t test. Mann–Whitney U test and Kruskal–Wallis test were used, respectively, to compare means of 2 and 3 or more groups of variables not normally distributed. The frequencies of categorical variables were compared using Pearson χA total of 480 patients were enrolled in the five-year study period, including 265 (55.2%) males and 215 (44.8%) females. The median age was 72.5  and there were 145 (30.2%) smokers. Lymph node metastasis was observed in 321 (66.9%) patients and TNM stage ranged from I–II  and III–IV . By the end of study, 345 (71.9%) patients survived and 135 (28.1%) patients died .mRNA levels of Beclin 1 were compared in 50 tissue pairs of normal tumor adjacent and corresponding tumor tissue and the results showed that Beclin 1 mRNA levels were significantly higher in the tumor tissues than in the normal tumor adjacent tissues (The Kaplan–Meier (KM) method was used to analyze time-to- all-cause mortality for patients with high and low Beclin 1 expression, and negative and positive EGFR mutations and ALK rearrangements. As shown in Fig. To assess the factors associated with PFS, Beclin 1 level, EGFR and ALK mutations, TNM stage, differentiation and lymph node metastasis were included in the Cox regression models as independent variables. The results showed that Beclin 1 (OR = 2.882), EGFR (OR = 1.672), ALK (OR = 6.982) mutations and tumor differentiation grade (OR = 2.212), TNM stage (OR = 2.772) and lymph node metastasis (OR = 6.222) were independently associated with PFS in the patients with NSCLS Table , and higP = 0.018), 0.781 and 15% (P = 0.011) and 0.722 and 11% (P = 0.010), respectively  and better predict the prognosis for developing targeted treatment plan and improve the efficiency of diagnosis and treatment and reduce the risk of death . At presBeclin 1 is an autophagy-related gene and it interacts with Bcl-2 to induce apoptosis via binding to Bcl-2 and Bcl-xL, followed by the release of cytochrome c into the cytosol and activation of caspases . The pro47. In the non-squamous NSCLC, the role of EGFR mutations on of lymph node metastasis have not been fully investigated and more works are needed to elucidate the molecular mechanisms.EGFR, as a member of the receptor tyrosine kinase family, is closely associated with the occurrence and development of NSCLC , ovarianALK gene rearrangements are present in a small subset of NSCLC . In thisOur analysis also showed that Beclin 1, EGFR and ALK are independently associated with PFS and are significant predictors for PFS in the patient with non-squamous NSCLC with AUCs above 0.70, suggesting that they may be used as standing alone biomarkers or in combination with other diagnostic tools to precisely predict the outcome of therapeutic plan. Since these markers can be assessed using biopsy samples or surgically resected samples, the results could be available at the early stage of treatment and would be used to stratify patients for optimal chemotherapy options including the use of immune checkpoint inhibitors , 62.There are several important limiting points in this study: it is its retrospective nature and single center-study with limited number of participants, patients were limited to elderly, were followed-up for relative short time and were not subjected to the same surgical treatments. Large and perspective studies are needed to further validate our conclusions.Non-squamous NSCLC is one of the most aggressive subtypes of lung cancer. Beclin 1 expression, EGFR and ALK mutations are independently associated with the prognosis and have prognostic value to predict PFS in the patient with non-squamous NSCLC. Therefore, Beclin 1, EGFR and ALK may be used as standing alone biomarkers or in combination with other diagnostic tools to predict the outcome of therapeutic plan and stratify patients for better prognosis.


All animals developed septic shock after a median of 15 (14–19) h. When MAP was raised using norepinephrine, the PbtO2 increased significantly (from 41 ± 4 to 55 ± 5 mmHg), and the cerebral lactate/pyruvate ratio decreased (from 47 ± 13 to 28 ± 4) compared with values at shock onset. Changes in the microcirculation were unchanged with restoration of MAP and the glutamate increased further (from 17 ± 11 to 23 ± 16 μM), as it did in the untreated animals. In septic shock, the correction of hypotension with vasopressors may improve cerebral oxygenation but does not reverse the alterations in brain microcirculation or cerebral metabolism.The effects of reversal of hypotension on the cerebral microcirculation, oxygenation, and metabolism in septic shock remain unclear. In 12 sheep, peritonitis was induced by injection of feces into the abdominal cavity. At the onset of septic shock  < 65 mmHg, unresponsive to fluid challenge), a norepinephrine infusion was titrated in eight sheep to restore a MAP ≥ 75 mmHg; the other four sheep were kept hypotensive. The microcirculation of the cerebral cortex was evaluated using side-stream dark-field video-microscopy. Brain partial pressure of oxygen (PbtO Sepsis still represents a major health issue, with a persistently high mortality rate, despite considerable progress in diagnosis and therapy . AssociaBrain dysfunction is common in septic patients and is associated with poor outcome . AlthougThe study protocol was approved by the Institutional Review Board for Animal Care of the Free University of Brussels, Brussels, Belgium. Care and handling of the animals followed National Institutes of Health guidelines.2O, an inspired oxygen fraction (FiO2) of 1, an inspiratory time to expiratory time ratio of 1:2, and a square-wave pattern. The respiratory rate was adjusted to maintain an end-tidal carbon dioxide pressure  between 35 and 45 mmHg before arterial cannulation was performed. A 60 cm plastic tube (inner-diameter 1.8 cm) was inserted into the stomach to drain its content and to prevent rumen distension. A 14F Foley catheter  was placed in the bladder to collect the urine output throughout the experiment.Twelve female sheep, weighing between 27 and 35 kg, were fasted for 24 h with free access to water prior to the experiment. On the day of the experiment, the animals were premedicated using intramuscular midazolam  and ketamine hydrochloride  and then placed in the supine position. A peripheral venous 18-gauge catheter  was used to cannulate the cephalic vein and fentanyl citrate  and rocuronium bromide  were administered intravenously for endotracheal intubation . All sheep were thereafter sedated with a continuous intravenous administration of midazolam (0.2 mg/kg*h), ketamine hydrochloride (0.5 mg/kg*h), and morphine (0.2 mg/kg*h), and muscular blockade was given  throughout the experiment to avoid movement artefacts. Boluses of fentanyl (5 mg) were administered if needed in case of tachycardia and/or hypertension suggesting insufficient anesthesia. Mechanical ventilation  was initiated using the following settings: a tidal volume of 10 mL/kg, a respiratory rate of 12–16 breaths/min, a positive end-expiratory pressure (PEEP) of 5 cm HThe right femoral artery and vein were surgically exposed. A 6F arterial catheter  was introduced into the femoral artery and connected to a pressure transducer  zeroed at mid-chest level. An introducer was inserted through the femoral vein, and a 7F pulmonary artery catheter  was advanced into the pulmonary artery. A midline laparotomy was then performed. After surgical opening of the cecum, 1.5 g/kg body weight of feces was collected; the cecum was then closed, and the area around the cut was disinfected with iodine solution. An additional suture was inserted to prevent contamination and the cecum was returned to the abdominal cavity. A large plastic tube was inserted through the abdominal wall for later injection of feces. The abdomen was then closed in two layers. During the surgical operation, Ringer’s lactate and 6% hydroxyethyl starch (HES) solutions were infused at rates of 1 mL/kg*h and 2 mL/kg*h, respectively. After abdominal surgery, the animals were turned to the prone position and allowed to stabilize. A bilateral craniotomy was performed in all animals using a high-speed drill and a fine wire saw ; two holes were opened and connected until a segment of bone (bone flap) of about 3 × 3 cm was created in the sheep’s frontal bone, as previously described . The dur2 between 80 and 120 mmHg and PaCO2 between 35 and 45 mmHg, according to repeated hourly blood gas analysis . Hemoglobin concentration and oxygen saturation were measured with an analyzer calibrated for ruminant animals . Peak airway pressure, plateau airway pressure, expiratory gas flow, and FiO2 were recorded hourly. The total amount of blood withdrawn for gas analyses was estimated at around 60 mL . All monitored variables were recorded every 60 min. Measurements of mean arterial pressure (MAP), pulmonary arterial pressure, right atrial pressure, and pulmonary artery occlusion pressure (PAOP) were obtained at end-expiration . Core temperature and cardiac output  were monitored continuously. Body surface area, cardiac index, stroke volume index, and systemic vascular resistance (SVR) were calculated using standard formulas.Mechanical ventilation parameters were adjusted to keep PaOThe microvascular network of the cerebral cortex was visualized on the left lobe using an SDF video-microscopy system  with a 5× imaging objective giving 326× magnification. The lens of the imaging device was covered with a disposable sterile cap and was applied without pressure to the cerebral frontal cortex. Because of brain pulsatility, this was best accomplished by placing the device on a metallic arm for stabilization . Lack of pressure was ensured by preservation of flow in large vessels . At leas2) catheter was introduced through the right cranial hole and connected to a specific monitor , in which the temperature was manually adjusted to blood temperature. Probe function was confirmed by an oxygen challenge (FiO2 1.0 for 5 min). The CMA 20 catheter was introduced close to the PbtO2 catheter and perfused by a miniaturized infusion pump  with a CNS perfusion fluid  at a flow rate of 1.0 μL/min. This flow rate guarantees an almost 50% recovery rate for molecules of less than 20 kDa and was selected to provide additional fluid for further research on brain metabolites. To allow for probe equilibration, data from the first hour after placement were not used. After one hour of stabilization, the perfusate was collected every 60 min in specific microvials. Samples were analyzed for lactate, pyruvate, glycerol, glutamate, and glucose using an automatic analyzer . The lactate/pyruvate ratio (LPR) was automatically calculated.An intracranial brain oxygen pressure  < 65 mmHg, unresponsive to fluid challenge), norepinephrine was administered in 8 sheep and titrated to restore a MAP ≥ 75 mmHg (NE group); the 4 other sheep were kept hypotensive (No-NE group). Measurements of all hemodynamic and cerebral variables were obtained at baseline, 6 h and 12 h, at shock onset, and 2 h after the start of norepinephrine infusion.After the surgical procedures, baseline measurements, including cerebral microcirculation, oxygenation, and metabolism, were obtained. Feces were then injected into the abdominal cavity (sepsis group). Ringer’s lactate solution (RL) and 6% hydroxyethyl starch solution  were titrated to prevent hypovolemia, i.e., to maintain the PAOP at baseline levels, as previously reported . Moreovep value < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 24.0 for Windows . Data are presented as mean ± SD or median (ranges), as appropriate. The significance of differences in the measured variables between the different time-points was analyzed using a mixed-effects model for repeated measures, followed by a Sidak post hoc correction. A All animals developed septic shock after a median time of 15 (14–19) h after feces injection. The main hemodynamic, respiratory, and metabolic variables are shown in MAP was higher and lactate levels lower after 2 h of norepinephrine administration in the NE group compared with the untreated animals. All other variables were similar between the two groups at the different time-points.2 decreased significantly, and brain metabolism deteriorated; in particular, the LPR increased, as did glutamate and glycerol  alterations in cortical cerebral microcirculation cannot be reversed by the administration of norepinephrine; (b) the reduction in brain oxygenation and the increase in LPR, as markers of tissue hypoxia and anaerobic metabolism, respectively, are partially reversed by the correction of hypotension, although they remain below baseline and physiological values; (c) persisting increases in cerebral glycerol or glutamate, suggesting ongoing excitotoxicity and cellular damage, are unresponsive to the reversal of hypotension using norepinephrine.In this study, the septic animals had already undergone initial fluid resuscitation and had been in shock for two hours; no other changes in treatment were made after vasopressor initiation to ensure that alterations in the measured variables were not influenced by factors other than the increase in MAP. Norepinephrine is presently considered as the vasopressor of choice in septic shock ; moreoveHow can one explain the lack of an effect of effective vasopressor support on the cerebral microcirculation? As tissue perfusion and microvascular flow depend, among other factors, on the diameter of the vessel and the applied pressure gradient, the vasoconstricting effects of norepinephrine may increase the cerebrovascular resistance  and this may limit the increase in microvascular flow induced by the increase in perfusion pressure. Another potential explanation is that, if cerebral blood flow autoregulation was preserved, a regional microvascular response may have occurred to maintain constant brain perfusion over time, thus resulting in no change in the vascular tone or in the microcirculatory flow . HoweverEscherichia coli for 24 h was associated with a progressive decrease in MAP and PaO2 over time, which was associated with a significant reduction in PbtO2 (from 32.2 ± 10.1 to 18.8 ± 11.7 mmHg after 3 h and to 22.8 ± 5.2 mmHg after 24 h of sepsis) [2 observed in this study might have been secondary to hypotension  and hypoxemia , which can be easily corrected in clinical practice. In our experimental model, as PbtO2 changes could not be entirely restored to baseline values but remained significantly higher in animals treated with norepinephrine compared with others, we could argue that the reduction in cerebral oxygenation during sepsis could be initially related to early microvascular alterations and further aggravated by systemic hemodynamic factors, such as hypotension; although hypotension and related tissue hypoxia could be easily reversed by the administration of vasopressors, persistent microvascular alterations contributed to maintain lower cerebral oxygenation values than initial values. Importantly, cerebral microcirculation was assessed at the pial level, while the PbtO2 catheter generally assesses oxygenation within the gray matter; regional blood flow to these areas may be dissociated in shock, at least in ischemia/reperfusion models [Brain hypoxia has already been observed in experimental sepsis . In anot sepsis) . HoweverThe switch toward anaerobic metabolism, as indicated by an increased LPR, was also partially, but not entirely, reversed by norepinephrine. Cerebral metabolic derangements are poorly characterized during sepsis. No changes in brain metabolism over time were observed in another swine model of resuscitated sepsis ; howeverThere are some limitations to this study. First, we visualized only pial vessels and the frontal cortex, and these areas may not be representative of deeper brain structures, including white matter and the brain stem. Second, we did not measure regional CBF, flow autoregulation, or cellular function, and our findings cannot be correlated with cerebral perfusion or neuronal electrical activity. Third, we did not perform any post mortem histological examination of brain parenchyma. Fourth, our model is lethal and we could not evaluate any association between microvascular disturbances and clinical neurological abnormalities. Fifth, we did not study different levels of MAP and their relative effects on cerebral microvascular perfusion. Sixth, source control and antibiotics were not provided as suggested to increase the translatability of research findings on animal models of sepsis ; howeverIn this ovine model of sepsis, reversal of hypotension using norepinephrine resulted in partial correction of tissue hypoxia and did not influence anaerobic metabolism. Persistent alterations of the cerebral microcirculation may explain these findings.


To investigate the incidence of frozen shoulder and risk factors for the onset of frozen shoulder in middle-aged and elderly subjects within 1 year of discharge from a hospitalization that involved intravenous infusion in Zhangjiagang Second People's Hospital.A total of 1,900 subjects who were discharged from a hospitalization that involved intravenous infusion in the hospital between May 2020 and September 2020 met the inclusion criteria for this study: 950 subjects had a mean daily duration of intravenous infusion ≤ 2 h (low exposure) and 950 subjects had a mean daily duration of intravenous infusion ≥3 h (high exposure). Subjects were followed up by telephone at 6 months ± 1 week and 12 months ± 1 week after discharge the incidence of frozen shoulder.P < 0.001); subjects hospitalized for 11–30 days had a higher risk of frozen shoulder compared to those hospitalized for 10 days or less ; subjects who were overweight/ obese (BMI ≥ 25 kg/m2) had a higher risk of frozen shoulder compared to those of normal weight (BMI 18.5–24.9 kg/m2) ; subjects in the 56–70-year-old age group had a higher risk of developing frozen shoulder compared to those in the 40–55-year-old age group ; diabetes increased the risk of frozen shoulder . The 71–85 years old age group and hypertension were statistically significant in univariate analysis but not in multivariate analysis (P > 0.05).The cumulative incidence rate of frozen shoulder within 1 year of discharge was 5.2%. Multivariate logistic regression analysis revealed the risk of frozen shoulder was higher in subjects with a mean daily duration of intravenous infusion ≥3 h compared to ≤ 2 h (OR = 3.082, 95% CI 1.919–4.949, 2, age 56–70 years, and diabetes.Compared with middle-aged and elderly in the general population, middle-aged and elderly subjects who received intravenous infusion during a hospitalization had a higher cumulative incidence rate of frozen shoulder within 1 year after discharge. Independent risk factors for the onset of frozen shoulder included mean daily duration of intravenous infusion ≥3 h, length of hospital stay 11–30 days, BMI ≥ 25 kg/m Frozen shoulder is a common clinical disorder. In addition to pain, frozen shoulder can cause a gradual reduction in the range of motion of the shoulder joint, which seriously impacts all areas of an affected individual's work and life. Globally, the incidence of frozen shoulder is estimated at 2–5% . In 1934Frozen shoulder is a self-limiting condition. The course of disease is generally between 2 and 3 years, but 50–70% of patients will have varying degrees of shoulder pain and limited mobility for a longer period of time . Common The etiology of the frozen shoulder has not been fully understood. The Upper Limb Committee of the International Society of Arthroscopy, Knee Surgery and Orthopedic Sports Medicine (ISAKOS) classify frozen shoulder as primary (idiopathic) or secondary  , 9, 10. In our acupuncture department, we treat a large number of patients with frozen shoulder each year. While obtaining routine medical history from our patients, we found that some patients had been hospitalized and received intravenous infusion prior to experience frozen shoulder, and they had shoulder pain and limited mobility for some time after discharge without obvious predisposing factors. A previous report identified prolonged post-operative intravenous infusion as a risk factor for frozen shoulder in patients who underwent neurosurgery . Previouhttp://www.chictr.org.cn; registration number: ChiCTR2000031862). In this study, the exposure was intravenous infusion, which uses the principles of atmospheric pressure and hydrostatic pressure to infuse a large amount of sterile fluid, electrolytes, and/or drugs into the body. The outcome was incidence of frozen shoulder. The mean daily duration of intravenous infusion was calculated as total duration of intravenous infusion during hospitalization divided by the number of hospitalization days. According to our previous unpublished study, subjects were stratified based on mean daily duration of intravenous infusion as high exposure (mean daily duration of intravenous infusion ≥3 h) or low exposure (mean daily duration of intravenous infusion ≤ 2 h).This single-center, prospective cohort study was conducted in compliance with the Declaration of Helsinki . The resn = 862. Based on clinical experience, we expected a 10% drop-out rate; therefore, the effective sample size for each group was calculated as n = 950.PASS 11 software was used to estimate the sample size according to the relative risk (RR) of the outcome of the two groups of subjects that underwent intravenous infusion. We conducted a preliminary study in middle-aged and elderly subjects who were discharged from a hospitalization that involved intravenous infusion. Findings showed that the incidence of frozen shoulder was 6.7% in the high-exposure group and 3.3% in the low-exposure group, with an RR of 2.03. The present study included an equal number of subjects in the low and high exposure groups. With α = 0.05 and power 1-β = 0.9, the effective sample size for each group was calculated as Subjects who were discharged from a hospitalization that involved intravenous infusion in the Second People's Hospital of Zhangjiagang City, Jiangsu Province, China between May 2020 and September 2020 were eligible for this study. Inclusion criteria were: (1) subjects discharged from a hospitalization that involved intravenous infusion; (2) subjects aged 40–85 years; (3) subjects hospitalized for 1–30 days; (4) subjects signed an informed consent form upon discharge and agreed to post-discharge follow up. Exclusion criteria were: (1) subjects aged <40 years or >85 years; (2) subjects hospitalized for >30 days; (3) subjects admitted to hospital due to trauma-induced shoulder pain and dysfunction, such as shoulder sprain, fracture, dislocation, rupture of the supraspinatus tendon, etc. (4) subjects who had frozen shoulder before admission and had recovered, or had frozen shoulder at admission and during hospitalization; (5) subjects who had undergone craniocerebral or other neurosurgical procedures before admission or during hospitalization, or suffered from intracranial lesions etc., and had poor recovery after discharge, resulting in decreased muscle strength of the neck and shoulder (muscle strength ≤ grade 4), causing limited activity of the shoulder joint; (6) subjects who underwent surgery on the shoulder, neck, chest, and other parts that would later lead to stiffness and adhesion of the soft tissue around the shoulder joint; (7) subjects with mental disorders, such as dementia, psychiatric diagnosis, and intellectual retardation etc, or who could not clearly express themselves for other reasons; (8) subjects who were unwilling to sign the informed consent form or expressly stated that they would not comply with follow-up.The primary endpoint was the incidence of frozen shoulder within 1 year of discharge from a hospitalization that involved intravenous infusion. The secondary endpoint was the identification of risk factors for the onset of frozen shoulder following discharge from a hospitalization that involved intravenous infusion.We included subjects in order of discharge until we met the required sample size for each group. During the period we included a total of 1,900 subjects meeting the criteria. There were 950 cases in each group of exposure group and control group. The hospital records of included subjects were reviewed. Demographic characteristics  and clinical characteristics  were recorded.Follow-up was calculated from the day of hospital discharge. Subjects were followed-up by telephone at 6 months ± 1 week and 12 months ± 1 week after hospital discharge by specially trained staff. Subjects were asked whether they had shoulder pain and limited mobility. Subjects who reported shoulder pain and limitation of activities were asked whether there were any predisposing factors such as shoulder joint trauma or central and peripheral nerve injury. Subjects with no predisposing factors were instructed to attend the hospital for a follow-up visit, where frozen shoulder was diagnosed based on criteria reported in the literature , 14, 15.At the follow-up hospital visit, an experienced orthopedic surgeon asked the subjects about their medical history and conducted a physical examination. X-ray, MRI, or other examinations may have been performed to exclude other joint and soft tissue disorders such as acromioclavicular arthritis, rheumatoid arthritis, septic arthritis, supraspinatus tendonitis, subacromial bursitis or biceps long head tendonitis; bone structural abnormalities such as osteonecrosis, primary and metastatic tumors or Paget's disease; neck lesions such as cervical spondylosis or thoracic outlet syndrome; or shoulder pain caused by visceral lesions such as upper lung tumor, esophagitis, myocardial infarction, digestive tract ulcers, or cholecystitis. This procedure screened out subjects who did not meet the diagnostic criteria for frozen shoulder, those with frozen shoulder were included in the analysis.2 test. Rate difference and corresponding 95% confidence intervals (95% CIs) were calculated with the Wilson method. The associations between relevant risk factors and the onset of frozen shoulder were explored with binary logistic regression analysis. The dependent variable, frozen shoulder, was binary , and the independent variables were binary or ordered multi-category. Binary variables were: mean daily duration of intravenous infusion , gender , work status , hypertension , diabetes , surgical history , and length of hospital stay . Ordered multi-category variables were: age , BMI , and education . Potential risk factors were identified with univariate analysis. Factors with P < 0.05 were recruited into multivariate logistic regression analysis. Odds ratios (OR) with 95% CIs were calculated. P < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS 21.0 software. Multiple imputation was used to handle missing data . CategorP = 0.680)  and 950 subjects with a mean daily duration of intravenous infusion ≥3 h (high exposure). Overall, 78 (8.2%) subjects with a mean daily duration of intravenous infusion ≤ 2 h (low exposure) and 83 (8.7%) subjects with a mean daily duration of intravenous infusion ≥3 h (high exposure) were lost to follow-up, with no significant difference in lost to follow-up rates between subjects with low and high exposure to intravenous infusion  . SubjectDuring follow-up, a total of 98 subjects had frozen shoulders, for a cumulative incidence rate of 5.2%: 31 subjects with a mean daily duration of intravenous infusion ≤ 2 h were diagnosed with frozen shoulder, for a cumulative incidence rate of 3.3%; 67 subjects with a mean daily duration of intravenous infusion ≥3 h were diagnosed with frozen shoulder, for a cumulative incidence rate of 7.1% (RR = 2.15).n = 29, mean daily duration of intravenous infusion ≤ 2 h; n = 50 mean daily duration of intravenous infusion ≥3 h), and 65 subjects were diagnosed with frozen shoulder , for an incidence rate of 3.4%, and accounting for 66.3% of the cumulative cases.At the first follow-up, 79 subjects reported shoulder pain , and 33 subjects were diagnosed with frozen shoulder , accounting for 33.7% of the cumulative cases.At the last follow-up, 64 subjects reported shoulder pain (P < 0.05). There were no significant associations between subjects' gender, education, work status, and surgery and the onset of frozen shoulder . The model could correctly classify 95.2% of the predicted values. The model sensitivity was 6.1%, specificity was 100%, positive predictive value was 100%, and negative predictive value was 95.1%.The Hosmer-Lemeshow goodness of fit index for the multivariate logistic regression model was good (P < 0.001); subjects hospitalized for 11–30 days had a higher risk of frozen shoulder compared to those hospitalized for 10 days or less ; subjects who were overweight/ obese (BMI ≥ 25 kg/m2) had a higher risk of frozen shoulder compared to those of normal weight (BMI 18.5–24.9 kg/m2) ; subjects in the 56–70-year-old age group had a higher risk of developing frozen shoulder compared to those in the 40–55-year-old age group ; diabetes increased the risk of frozen shoulder . The 71–85 years old age group and hypertension were statistically significant in univariate analysis but not in multivariate analysis (P > 0.05)  .2, age 56–70 years, and diabetes.This study investigated the incidence of frozen shoulder and risk factors for the onset of frozen shoulder in middle-aged and elderly subjects within 1 year of discharge from a hospitalization that involved intravenous infusion. Findings showed the cumulative incidence rate of frozen shoulder within 1 year of discharge was 5.2%, with frozen shoulder within 6 months of discharge accounting for 66.3% of the cumulative cases. Independent risk factors for the onset of frozen shoulder in middle-aged and elderly subjects who were discharged from a hospitalization that involved intravenous infusion included mean daily duration of intravenous infusion ≥3 h, length of hospital stay 11–30 days, BMI ≥ 25 kg/mThe incidence of frozen shoulder among the middle-aged and elderly subjects included in this study was higher than previously reported for the general population. In Europe and the US, frozen shoulder affects an estimated 2% of the general population, with a cumulative incidence of 2.4 cases per 1,000 person-years ; in the In the present study, mean daily duration of intravenous infusion and length of hospital stay were independent risk factors for the onset of frozen shoulder in middle-aged and elderly subjects within 1 year of discharge from a hospitalization that involved intravenous infusion. Patients must temporarily limit upper extremity activities during intravenous infusion. A long duration of intravenous infusion and prolonged hospital stay will cause substantial limitations on upper extremity activities, increasing the incidence of frozen shoulder. A study in patients who had surgical treatment for sub-arachnoid hemorrhage reported the incidence of frozen shoulder at 6 months of follow-up was 25.3%, and the development of frozen shoulder was associated with duration of post-operative intravenous infusion . ConsistP = 0.009); however, hypertension was not an independent risk factor for frozen shoulder on multivariate analysis (P = 0.862). Evidence suggests that the incidence of hypertension increases with age and in subjects with diabetes, implying an interaction between hypertension and age and diabetes. Accordingly, we performed ad-hoc analyses. Consistency testing of our data using SPSS 21.0 software showed that the OR values for hypertension and frozen shoulder in subjects aged 40–55, 56–70, and 71–85 years, were 1.775, 1.711, and 1.144, and the incidence of frozen shoulder tended to decrease with increasing age, indicating that age was an effect modifier for hypertension. The OR values for hypertension and frozen shoulder in subjects with no diabetes or diabetes were 1.445 and 0.856, respectively, which indicated that the pathogenesis of hypertension on frozen shoulder was affected by diabetes.Accumulating evidence suggests that diabetes and hypertension are risk factors for the onset of frozen shoulder , 23, 24.Some studies showed no association between obesity and the incidence of frozen shoulder , 28. OthMany studies , 30 haveP = 0.681). This may be because our study was limited to a specific population of subjects that received intravenous infusion during a hospitalization, gender-specific differences in the onset of frozen shoulder have been influenced by the effect of intravenous infusion or other confounders.Previous studies showed frozen shoulder is more common in women than men, with women comprising an estimated 58.0–60.9% of subjects with frozen shoulder , 35. Raw(1) This study only analyzed subjects receiving inpatient intravenous infusion in a single center and the study sample was limited by conditions, which affected the extrapolation of the study results. (2) The sample size was small and the incidence of frozen shoulder in the study population was low, resulting in a relatively small number of total cases. (3) The baseline characteristics of subjects with a mean daily duration of intravenous infusion ≤ 2 h (low exposure) and a mean daily duration of intravenous infusion ≥3 h (high exposure) were not matched using propensity scores; therefore, our findings may have been influenced by cofounders. (4) Our analyses did not consider mean daily durations of intravenous infusion between 2 and 3 h.2, age 56–70 years, and diabetes. In middle-aged and elderly subjects that undergo intravenous infusion during hospitalization, the incidence of frozen shoulder after discharge may be reduced by accurate identification of these risk factors and timely intervention with appropriate functional exercises.Compared with middle-aged and elderly subjects in the general population, middle-aged, and elderly subjects who received intravenous infusion during a hospitalization had a higher cumulative incidence rate of frozen shoulder within 1 year after discharge, and most incidences of frozen shoulder occurred within 6 months after discharge. Risk factors for the onset of frozen shoulder in middle-aged and elderly subjects discharged from a hospitalization that involved intravenous infusion were mean daily duration of intravenous infusion ≥3 h, length of hospital stay 11–30 days, BMI ≥ 25 kg/mhttp://www.medresman.org.cn).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Clinical trial public platform management (The studies involving human participants were reviewed and approved by Ethics Committee of Zhangjiagang Second People's Hospital. The patients/participants provided their written informed consent to participate in this study.YC is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. WC conceived and designed the study and wrote the manuscript. JC performed the statistical analysis. JP and YF carried out the literature search and data collection. WC and JC conducted an investigation. YC and JC revised the manuscript. All authors contributed to the article and approved the submitted version.This research was financed by science and technology support plan of Zhangjiagang City in 2020 [Grant No. ZKS2027] and Zhangjiagang City Health Youth Science and Technology Projects in 2020 [Grant No. ZJGQNKJ202037].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.