The aim of the present study was to assess the prevalence of deficient mismatch repair (MMR) in Chinese ovarian clear cell carcinoma (CCC) patients and its association with clinicopathologic features.Immunohistochemistry with four antibodies against MLH1, PMS2, MSH2 and MSH6 was performed on whole section slides, and the results were correlated with clinicopathologic variables.n = 4) and MLH1/PMS2 (n = 2). The average age of the six patients with deficient MMR was 45.6 years, and the rate of MMR-deficient tumors in women ≤50 years was relatively higher than that in women over 50 years . Half of the patients with deficient MMR were diagnosed with synchronous  and metachronous  cancer, which was significantly more than their intact counterparts (P = 0.002). All six patients with deficient MMR had early-stage tumors, and the majority (83.3%) were platinum sensitive. The median progression-free survival was slightly higher in patients with defective MMR expression than in their intact counterparts (30 months vs. 27 months), but significance was not achieved (P = 0.471).A total of 108 cases were included in the present study with a median age of 52 years at first diagnosis. Early-stage disease and platinum-sensitive recurrence accounted for 62.3 and 69.6%, respectively, of the total cases. Overall, the estimated 5-year overall survival was 70.3 and 20.7% in patients with early- and late-stage tumors, respectively. Deficient MMR was identified in 5.6% (6/108) of the cohort and included MSH2/MSH6 (Young ovarian CCC patients with concurrent diagnosis of endometrial and colorectal cancer are more likely to have MMR-deficient tumors, thereby warranting additional studies to determine whether patients harboring MMR abnormalities have a favorable prognosis.The online version contains supplementary material available at 10.1186/s12885-022-09588-z. The histological subtypes of ovarian cancer are distinct diseases, each with different clinical and molecular characteristics . OvarianMismatch repair (MMR) deficiency has been demonstrated to be a biomarker of sensitivity to immune checkpoint blockade with antibodies against programmed death receptor-1 across various types of tumors . DefectiIn the present study, we assessed the status of MMR proteins in a well-annotated unselected cohort of Chinese ovarian CCC patients. The frequency of MMR deficiency was evaluated by immunohistochemistry, and the association of MMR deficiency with clinicopathologic variables was also evaluated.All methods were carried out in accordance with relevant guidelines and regulations. After obtaining approval from the Institutional Review Board (050432–4-1212B), we identified all the patients by searching the surgical pathology archives for “ovarian clear cell carcinoma” from 2008 to 2018. In our institution, one surgical specimen is generally reviewed by two pathologists (one young and one senior doctor) as a routine. A third experienced pathologist will review the slides in some difficult cases or resolve discrepancies. In the present study, we included all patients with archived tissue blocks, and all available hematoxylin & eosin-stained slides were reviewed to confirm the diagnosis. Cases were excluded if they were focal carcinomas or if the clear cell component was less than 50% in mixed tumors. The requirement for written informed consent was waived considering the retrospective design of the study.Clinicopathological information and survival outcomes were obtained from medical records. The following data were collected: the age at diagnosis of ovarian CCC; personal and family history of cancer; date and type of primary surgery; International Federation of Gynecology and Obstetrics (FIGO) stage at initial diagnosis ; consistVentana BenchMark ULTRA) according to our protocols as MMR protein immunohistochemistry is routinely conducted at our institution [Four-micrometer-thick, formalin-fixed, paraffin-embedded whole-block sections were used for immunohistochemistry. All staining was performed using an automated slide stainer  and normal (retained nuclear staining) , 22. Lymt tests were utilized to evaluate continuous variables, while chi-square tests (continuity correction chi-square) were used to evaluate categorical variables. Survival time was evaluated using the Kaplan–Meier model. All reported P values were two-sided, and P < 0.05 was considered statistically significant. Statistical Package for Social Science (SPSS)  was used for the statistical analyses, and GraphPad Prism  was used to generate the figures.Continuous data are presented as the median/mean (range), and categorical data are presented as proportions. Parametric Student’s n = 3) or colon (n = 1). The patient with synchronous colon and ovarian cancer developed endometrial cancer 2 years later. A previous history of breast cancer and thyroid cancer was noted in two patients and one patient, respectively. The remaining two patients had metachronous urothelial cell cancer and lung cancer after the diagnosis of ovarian CCC. In addition, 23 patients reported a family history of cancer, mostly colorectal cancer (n = 4), pancreatic cancer (n = 3) and urothelial cell cancer (n = 3).In total, 108 patients were included in the present study after excluding 8 cases with immunohistochemistry technical failure. For the entire cohort, the median age was 52 years (mean of 51.8 years and range of 26–79 years), and 37.0% of the patients were 50 years or younger. Nine patients had a personal history of cancer and/or synchronous cancer. Of these patients, four were diagnosed synchronously with a malignancy of the endometrium (Table n = 4) and MLH1/PMS2 (n = 2) . In addition, the rate of MMR-deficient tumors in women ≤50 years was relatively higher than that in those over 50 years    patients harbored abnormal MMR expression, including MSH2/MSH6 (The two patients with a family history underwent subsequent genetic testing. Patient No. 6 was demonstrated to carry the following MHL1 germline mutation: c.1756G > C (p. Ala586Pro) . PatienP = 0.002), respectively  . After a mean follow-up time of 46 months , 46.1% of patients (48/104) died of disease, 20.2% of patients (21/104) were still alive with disease, and 33.7% of patients (35/104) had no evidence of disease. Of note, 26 patients had a follow-up time less than 24 months. Of these, six patients were alive without disease, and the remaining 20 patients all died. Figure P = 0.471) , but statistical significance was not achieved  has shown that patients with abnormal MMR expression are significantly younger with a mean age of 40 years compared to 53.2 years for the overall cohort [Universal testing of MMR in ovarian cancer is not routine in most hospitals, and the National Comprehensive Cancer Network (NCCN) guidelines recommend it as clinically indicated. Not surprisingly, higher frequencies of defective MMR have been reported in younger patients in several studies , 12, 24.l cohort . In the MMR-deficient tumors have peculiar clinical behaviors, including early-onset metastatic potential, but generally favorable prognosis and remarkable responses to immune therapy . The posTo the best of our knowledge, the present study represents one of the largest series measuring MMR proteins in ovarian clear cell carcinoma patients. However, the present study had several limitations. First, considering disease rarity, we collected the cases over a long period of time, which led to missing data. Second, the cohort may be limited by the selection and surveillance biases often associated with studies from a single institution. Last, the present study did not evaluate the specific regimen of treatment, which may be a confounding factor for survival outcome.The present study showed MMR loss in 5.6% of unselected tumors of ovarian clear cell carcinoma, but this rate increased to 10% when selecting for age (50 years and below). All patients presented with early-stage disease, and half of the patients had synchronous/metachronous endometrial/colorectal cancer. Patients with MMR deficiency seemed to have better progression-free survival when all patients were analyzed, but the difference was not as significance when only the early-stage group was investigated. Thus, additional studies are required to determine whether patients harboring MMR abnormalities have a favorable prognosis.Additional file 1: Supplementary Table 1. Clinical features of the study population (n = 108).


Acinetobacter baumannii (CRAB) bacteremia is a serious clinical problem worldwide. However, the factors related to the emergence and replacement of predominant CRAB clones in nosocomial settings are unclear. By multilocus sequence typing (MLST), we evaluated the genetic relatedness of CRAB bloodstream isolates at a tertiary care hospital over a 3.5-year period and investigated the clinical and microbiologic characteristics of the predominant sequence types (STs). One hundred and seventy-nine CRAB bloodstream isolates were collected from June 2016 to December 2019, and their MLSTs according to Oxford scheme and clinical data were obtained. The predominant STs were assessed for in vitro growth, competitive growth, and virulence in a mouse model of intraperitoneal infection. Two dominant clones—ST369 (n = 98) and ST191 (n = 48)—belonging to international clone 2 (IC2) were recovered from patients admitted to intensive care units (ICUs) or wards. ST191 predominated  from June 2016 to July 2017, whereas ST369 , which was first isolated from a patient admitted to the emergency room, replaced ST191  after August 2017. In a multivariate analysis, leukopenia  and ST191 or 369  were independent risk factors for 7-day mortality. Compared with non-ST369, ST369 was associated with a shorter time to bacteremia from ICU admission , pneumonia as an origin of bacteremia , leukopenia , and a lower 7-day survival rate . In vitro, ST 369 isolates had significantly higher growth rates and enhanced competitive growth compared to ST191. Finally, ST369 had greater virulence and a higher mortality rate than other STs in a mouse infection model. We report almost-complete replacement of the predominant ST191 clone by ST369 within an 8-month period at our hospital. ST369 had a high incidence density rate of CRAB bacteremia, a short time to bacteremia after ICU admission, and a high early mortality rate, which may be in part explained by its faster competitive growth rate and higher virulence than ST191.The clonal dissemination of carbapenem-resistant  Acinetobacter baumannii is an important cause of nosocomial infections, especially in the intensive care unit  is unprecedentedly high, which, combined with clonal transmission, has led to severe clonal outbreaks with high mortality rates in ICU settings  according to MLST using the Oxford scheme  were stored at −80°C. Species identification was performed initially by matrix-assisted laser desorption ionization-time of flight mass spectrometry-based VITEK MS  and VITEK 2 (bioMérieux) and was confirmed by using MLST-based identification as previously described  guidelines  stay before BSI within 2 months, time to BSI after ICU admission or hospitalization, presence of central venous catheter (CVC) within 48 h of BSI, early and late mortality, and appropriate antibiotics. The Charlson comorbidity index score was used to quantify the severity of pre-existing illness .A. baumannii was defined as A. baumannii resistant to both imipenem and meropenem by the broth microdilution test based on CLSI breakpoints  antakpoints ; (2) CRAakpoints . The minakpoints . Time toakpoints  on the fgltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD were sequenced  following the Oxford scheme . The houequenced . The isoin vitro growth rate was assessed for A. baumannii STs and the reference strain ATCC19606. ST191 and ST369 isolates were diluted to 1 × 106 CFU/ml after exponential growth in Luria-Bertani (LB) broth and incubated at 37°C with constant shaking at 180 rpm. At 12, 24, 36, and 48 h, a 10-fold serial dilution was spread on LB agar, and CFUs were enumerated after 24 h of incubation at 37°C. For each strain, three independent experiments were carried out at each time point.The In vitro competitive growth of ST369 and ST191 strains was assessed based on the competition index and percent recovery. Competitive growth of A. baumannii ST369 was assessed in a 1:1 mixture with A. baumannii ST191 strain by a PCR method  or gpi94 (ST191). The competition index was determined as follows: competition index = [CFU of isolates positive for gpi106 (ST 369) after 24 h cocultured ÷ CFU of isolates positive for gpi94 (ST191) after 24 h cocultured] ÷ [CFU of ST369 isolates inoculated ÷ CFU of ST191 isolates inoculated]. Three independent experiments were performed.R method . BrieflyA. baumannii strain, and randomly selected five gentamicin-resistant ST369 and five gentamicin-resistant ST191 A. baumannii isolates to create five combinations. Each set of bacterial isolates was separately cultured overnight in LB broth at 37°C and diluted 1:100. Gentamicin-resistant ST369 and ST191 were pooled with an equal number of the gentamicin-susceptible ST369 isolate, cocultured in LB broth at 37°C for 24 h, and serially diluted 1:10 in PBS. Next, 20 μl of each dilution were spread on LB agar and selective LB agar containing gentamicin (32 μg/ml). The CFU of the original suspensions was calculated by counting viable colonies on agar plates after overnight incubation at 37°C. For each combination, we determined the numbers of CFUs of (A) gentamicin-resistant ST369 and gentamicin-susceptible ST369 on LB agar, (B) gentamicin-resistant ST369 and gentamicin-susceptible ST369 on LB agar containing gentamicin, (C) gentamicin-resistant ST191 and gentamicin-susceptible ST369 on LB agar, and (D) gentamicin-resistant ST191 and gentamicin-susceptible ST369 on LB agar containing gentamicin. Percent recovery of ST369 was calculated as B ÷ A × 100 and of ST191 as D ÷ C × 100. The experiment was repeated three times for each set of combinations.Percent recovery was evaluated by modifying a method described previously . We usedA. baumannii ATCC19606 , ST369, ST191, and ST784 were used in the study. For intraperitoneal (ip) inoculation in mice, freshly cultured inocula were prepared from frozen stocks of A. baumannii as described previously  and eviously . Inoculat-test , Mann–Whitney test, or Kruskal–Wallis test  for continuous variables. Time-series count data corresponding to the incidence density of CRAB bacteremia were analyzed using a Poisson regression model. Growth was evaluated by paired t-test as described previously  and Prism  software.The Kolmogorov–Smirnov goodness-of-fit test was used to determine the normality of the data distribution. Categorical variables are expressed as percentages. Continuous variables are expressed as means ± SD if normally distributed and as medians and interquartile range  if non-normally distributed. In univariate analyses, Pearson’s chi-squared or Fisher’s exact test was used for comparisons of categorical variables and Student’s eviously . A KaplaA total of 179 bloodstream isolates were identified as CRAB by both MLST-based identification and resistance to carbapenem during the study period. MLST identified 11 distinct STs. ST369  was the most abundant ST, followed by ST191 , 784 , and ST451 . Using the default definition , nine STs  were assigned to IC2. Among 179 cases, most patients were in seven ICUs  at the onset of bacteremia, followed by a general ward  and the emergency room . ST369 was most abundant in the medical ICU , followed by ward , emergency room , and trauma ICU  during period from June 2016 to July 2017 (period 1), whereas ST369  replaced ST191  during period from August 2017 to December 2019 , minocycline , and colistin , and no isolate were susceptible to cephalosporin or quinolone . ST369 wThe sex ratio was 118:61  and the median age was 72  years. All CRAB bacteremia was healthcare associated and most patients  had one or more comorbidities. About half of patients were on mechanical ventilation  or had a CVC  prior to bacteremia. Most patients  had a history of ICU admission within 2 months prior to bacteremia. The most frequent source of bacteremia was pneumonia , followed by CLABSI  and skin and soft tissue infection . Adequate antibiotics were administered within 48 h in 62 patients 35%; .p = 0.07 by binary logistic regression]. As an origin of bacteremia, the frequency of pneumonia was higher in ST369 than non-ST369 . The incidence of leukopenia at the time of bacteremia was higher in patients with ST369 than non-ST369  and higher incidence of leukopenia . The 30-day survival rate of ST369 was 31% (30/96), similar to ST191 and ST784 . The median time to death after bacteremia onset was 4 , 12 , and 10  days for ST191, ST784, and other STs, respectively.The 7-day survival rate of CRAB bacteremia was 53% (92/172). The 7-day survival rate in patients with ST369 was significantly lower  than for ST191, ST784, and other STs  and higher Charlson comorbidity index score . Mechanical ventilator was more frequently used in fatal group . SOFA score and serum creatinine was higher in fatal group, compared to nonfatal group . In addition, leukopenia  and pneumonia  were more frequent in fatal group in univariate analysis. In fatal group, the proportion of CRAB bacteremia caused by ST191 or ST369 was higher than that of nonfatal group . In a multivariate analysis, Charlson comorbidity index score  and SOFA score , leukopenia , and CRAB bacteremia caused by ST191 or 369  were independent risk factors for 7-day mortality.We next evaluated risk factors for 7- and 30-day mortality in CRAB bacteremia . In 7-dap = 0.01], more underlying hypertension and diabetes  and higher Charlson comorbidity index score . The time interval from ICU admission to occur bacteremia was shorter in fatal group than nonfatal group . The proportion of mechanical ventilator and pneumonia was higher in fatal group . Fatal group had higher SOFA score and creatinine . Only an elevated SOFA score was an independent risk factor for 30-day mortality  in multivariate analysis  vs. 71  years, analysis .p < 0.05; Student’s t-test), indicating that ST369 had a higher growth rate than ST191  vs. 36% , p < 0.01; Mann–Whitney test; 5 CFU ST191, ST369, or ST784. After inoculation of 106 CFU, only mice in the ST369 group died within 48 h. After inoculation of 107 CFU ST191 or ST369, all mice died within 24 h but all mice inoculated with 107 CFU ST784 or ATCC19606 survived.We compared the survival rates of mice after ip inoculation with ST191, ST369, ST784, and ATCC19606 . All micAcinetobacter baumannii ST is an important determinant of the incidence of nosocomial CRAB bacteremia and of its severity and mortality. We report almost-complete replacement of the predominant ST191 clone as a cause of CRAB bacteremia by newly introduced ST369 within an 8-month period at our hospital; the incidence density rate of CRAB bacteremia increased thereafter. This observation was supported by the higher growth rate and competitive ability of ST369, compared to ST191 strains, as well as the hypervirulence of the former in a mouse model of infection. Our data highlight the need to be aware of the more rapid onset of ST369 CRAB bacteremia after ICU admission and its poor 7-day clinical outcomes, together with their greater potential for clonal spread.Although increased prevalence of CRAB has been reported in community-onset bacteremia without identifiable healthcare associated risk factors  CRAB is Some STs have high mortality rates in clinical settings . PatientA. baumannii from CRAB bacteremia are unclear. CRAB bacteremia caused by ST369 was associated with a shorter time to bacteremia from ICU admission (7 vs. 11 days), pneumonia as an origin of bacteremia (66 vs. 52%), and leukopenia (28 vs. 11%), compared to non-ST369 isolates. In addition, the 7-day mortality rate of patients with CRAB bacteremia caused by ST369 was significantly higher (59%) than for ST191 (42%) and other STs (30%). Acinetobacter baumannii virulence can be assessed by detecting virulence factors, such as the outer membrane protein OmpA, phospholipases, efflux pumps, penicillin-binding proteins, outer membrane vesicles . A waiver of consent was granted given the retrospective nature of the clinical analyses. Animal experiments were conducted in compliance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Chonnam National University and the Korean Food and Drug Administration. The study protocol was approved by the IACUC (CNU IACUC-H-2020-21) of Chonnam National University Hwasun Hospital.SK, JS, UK, and SI conceived and designed the study. YY, SS, and TO collected the clinical samples and data. SK and S-MC performed the experiments. SK, YY, SS, TO, and UK analyzed the data. SK, UK, S-JK, and K-HP contributed to data summary. SK, JS, UK, and SI contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Research Institute of Medical Sciences, Chonnam National University Medical School (2012-CURIMS-DR005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.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.


Sea spray aerosol (SSA) is a widely recognized important source of ice-nucleating particles (INPs) in the atmosphere. However, composition-specific identification, nucleation processes, and ice nucleation rates of SSA-INPs have not been well constrained. Microspectroscopic characterization of ambient and laboratory-generated SSA confirms that water-borne exudates from planktonic microorganisms composed of a mixture of proteinaceous and polysaccharidic compounds act as ice-nucleating agents (INAs). These data and data from previously published mesocosm and wave channel studies are subsequently used to further develop the stochastic freezing model (SFM) producing ice nucleation rate coefficients for SSA-INPs. The SFM simultaneously predicts immersion freezing and deposition and homogeneous ice nucleation by SSA particles under tropospheric conditions. Predicted INP concentrations agree with ambient and laboratory measurements. In addition, this holistic freezing model is independent of the source and exact composition of the SSA particles, making it well suited for implementation in cloud and climate models. Surface seawater marine microorganisms generate proteinaceous and polysaccharidic particulates that affect cold cloud formation. Sea spray aerosol (SSA) particles are emitted to the atmosphere from the ocean surface by wave breaking and bubble bursting  and, to a lesser extent, aspartic acid, an amino acid , or surface area and time . Application of ns for SSA particles has been successful in previous studies, e.g., where ns from intact and fragmented diatom cells , surface area . Last, we demonstrate the SFM approach for predicting atmospheric ice particle production from aqueous SSA and cloud droplets.In this study, we identified, using a controlled vapor cooling–stage microscope system, the components of SSA particles that nucleated ice with STXM/NEXAFS and CCSEM/EDX techniques . At T ≥ 220 K, IMF was observed for the same particles between RHice = 122 to 143%, which is likely a result of insoluble or solid biogenic INPs in the aqueous solution. Water uptake preceded IMF shown as open circles in ice compared to marine-derived particles. However, water uptake was not observed. It is likely that the examined continental particles contain less hygroscopic components compared to SSA or marine-derived particles . Those NEXAFS spectra are compared to spectra from bulk and SML water of unialgal and axenic cultures of T. pseudonana. INPs were typically large, with diameters ≥1 μm consisting of an inorganic core with an organic-rich exterior indicated by the blue and green colors, respectively. The corresponding spectra  averaged over these respective colored regions is also shown and highlights the similarity in absorption features of both blue and green spectra corresponding to carboxyl functionalities and CO3. Although inorganic compounds dominated the blue regions, OM still coats the particles. These functionalities were also observed for non–ice-nucleating SSA particles generated after 1 and 12 days of phytoplankton growth.X-ray spectromicroscopy and single-particle identification with assessment of NEXAFS features indicate the similarity in all mesocosm experiments between of the OM associated with aerosol-borne marine INPs, non–ice-nucleating SSA particles, and OM in seawater. T. pseudonana. In contrast, SSA particles from the Tpseu experiment showed only one specific compound. First, in the bulk and surface water of cultures, OM was present with two main absorption peaks similar to SSA particles and INPs, which we refer to as “type I” OM in Two distinct types of organic compounds were identified in cultures of N. atomus, E. huxleyi, and a natural mix of bacteria  are shown in figs. S3 to S8. Again, type I and II OM were found, but only type I OM was associated with INPs, SSA, and water. Alpert et al.  and may indicate unique acidic polysaccharide compounds. A similar beam damage effect was observed by Braun et al. , the ice spectrometer (IS), and the micro-orifice uniform deposit impactor–droplet freezing technique (MOUDI-DFT) (ns derived from SFM simulations are fit to previously reported [INP] and ns data from the LBWC and MART experiments (The SFM (UDI-DFT) . DetailsJhet values for IMF derived from all experimental data and our parameterization. Jhet is given as a function of Δaw (aw(T) of the aqueous solution droplet containing an INP (aw = 1 indicates pure water) and the ice melting curve, aw,ice(T), resulting in Δaw(T) = aw(T) − aw,ice(T). Note that Δaw = 0 would reflect freezing at the ice melting temperature at 273.15 K, which is not possible, and Δaw = 0.313 represents the homogeneous freezing limit for micrometer-sized cloud droplets  with parameters for IMF by SSA particles as m = 26.6132 and c = −3.9346. Jhet for SSA particles changed from 1.2 × 10−2 to 2.6 × 103 cm−2 s−1 between 265 and 240 K when aw = 1.0. This change in Jhet is similar for the range of Jhet determined for intact and fragmented phytoplankton cells  and pump at ground level. Aerosol particles were sampled approximately 20 m from breaking waves at these locations.k-means machine learning cluster analysis , hydroxyl functions (C─OH), ketone and carbonyl functions (C═O), carboxylic functions (COOH), and carbonate (CO3) mapped over individual particles. In addition, other elements such as K and Ca have absorption features at their L-edges near to the carbon K-edge and were identified. Damage to particles due to x-ray exposure was assessed by irradiating the same particle using a reduced x-ray dose multiple times and noting the absorption features that did or did not change as a result. Beam damage may alter intensities of peaks corresponding to C═C, COOH, and CO3 in individual particles. Therefore, we have used only the presence of absorption peaks and their peak x-ray energy position as a basis for comparison between the spectra.STXM/NEXAFS instrument at the 5.3.2 beamline of the Advanced Light Source was used to chemically map organic carbon functionalities (We identified single particles that acted as INPs using optical microscopy during ice nucleation experiments as described below and investigated these INPs using STXM/NEXAFS following a previous procedure (−1, and both ice formation and water uptake were observed. The frost point of the air particles were exposed to, Tfst, was first adjusted and set to remain constant in a range between 205 and 255 K. Ice nucleation experiments began when RHice = 90% by adjusting the particle T. Particles were cooled at a rate of 0.1 K min−1 while imaging all particles every 0.02 K (T = Tfst. This resulted in a small and consistent offset temperature correction between 0.1 and 0.3 K, which was applied to determine RHice. Calibration yielded experimental uncertainties of ΔT < ±0.3 K, ΔTfst < ±0.15 K, and ΔRHice < ±11% at 200 K and ±3% at 260 K. Multiple ice crystals nucleated at the same time in few instances, and these were considered as separate nucleation events. Frequently, ice continued to form after the first ice crystals were imaged; however, these were discarded since the water vapor pressure may not have been uniform when multiple ice crystals were present in the ice nucleation cell. In total, 68 ice nucleation events were recorded in this study.Ice nucleation and water uptake occurring below or at water saturation for SSA particles were optically imaged, which allowed for discrimination between DIN and IMF events (Jhet from our data and data from DeMott et al. (T and RHice at which ice nucleation occurs and the uncertainty of calculated Jhet and ns values due to repeating simulations in a Monte Carlo approach. DeMott et al. (et al. (Jhet corresponding to different particle types and different cooling rates and under different RH and T trajectories. This was the case when simulating IMF, DIN, and HIN shown in fig. S12.We used an SFM (


A. Baumannii is an opportunistic nosocomial pathogen which has severe antibiotic resistance. However, the epidemiology is less clearly understood in Jilin province and China. Thus, 89 A. baumannii isolates from a single hospital in Jilin province between 2013–2017 were performed by MLST. In order to better understanding of the epidemiology of Jilin isolates, Chinese strains originated from other domestic regions and worldwide isolates in MLST database were analyzed by silico phylogenetic tools together. A total of 22 STs in Jilin were identified, and 10 STs were found to be novel. The top three predominant sequence types are ST195 , ST208  and ST540 . ST369 is predicted to be group founder and ST195, ST540 are subgroup founders of the majority STs in Jilin Province. Some newly discovered singletons showed close relationship with strains from other countries, which suggest that nation-cross transmission is one of important origin of Jilin strains. The majority of Jilin STs showed clonality and close relationship with the majorities from other regions of China. But occupation of individual STs in Jilin were different from that of other domestic regions. The aggregation trend and genetic relationship proved that predominant Jilin STs continue to mutate during transmission. Drug resistance facilitated transmission of Jilin A.baumannii isolates because more than 94% of isolates are resistant to at least one carbapenem and the STs with strong resistance to carbapenems usually has more isolates. In conclusion, high diversity and different occupation of STs, and occupation of novel STs proved that epidemiology of A. baumannii in Jilin has special regional characteristics, and drug resistance facilitated transmission of domestic strains and foreign strains. A. baumannii in Jilin Province showed distinctive local characteristic.1. The epidemiology of A. baumannii.2. Newly discovered sequence types (STs), the results of genetic relatedness and geographical distribution of all STs showed epidemiological research by multi-locus sequence typing (MLST) in single hospital is helpful to monitor and control nosocomial infection of A. baumannii is about 94% in Jilin province (northeast China). Carbapenems resistance is a helpful factor for predominant STs spreading.3. The rate of carbapenems resistance among  A. baumannii is an opportunistic nosocomial pathogen inducing a vast array of infections, of which the most common infections are ventilator-associated pneumonia and bloodstream infections with high mortality rate [A. baumannii infections, carbapenem resistant A. baumannii has been prioritized as the top one of WHO priority pathogens list that are urgent to research and develop new antibiotics [The gram-negative bacterium ity rate . Multidribiotics , 3. Howeibiotics .A. baumannii, MLST helps not only in tracking the sequence types (STs) and outbreaks, but also in understanding the microbial evolution [MLST is a proven tool for the molecular typing of volution . The relvolution .A. baumannii is also on the rise, which has become a serious threat to public health [A. baumannii is less clearly understood in Jilin province as only six Jilin strains with same ST were registered in MLST database before our study. Therefore, STs of 89 A. baumannii isolates from a single hospital in Jilin between 2013–2017 were analyzed by MLST and other silico phylogenetic tools. For the sake of analyzing regional characteristics, previously registered Chinese isolates and large amount of global collection of A. baumannii from MLST database (https://pubmlst.org/organisms/acinetobacter-baumannii) were also covered in this study.In China, carbapenems resistant c health . Some pac health –10. HoweAmong obtained STs, the number of alleles in each locus varied from 5 to 14 and that of SNPs ranged from 5 to 63. The sequence diversity of each locus ranged from 2.98 to 6.40% with average value of 3.83%. Furthermore, nucleotide diversity (π) in this study was in the range of 0.00413 to 0.03514 (Table n = 81), 87.64% (n = 78), 89.89% (n = 80), 7.86% (n = 7), and 13.48% (n = 12), respectively. Twelve isolates were found to be sensitive to at least one of tested carbapenems. The overwhelming majority in our Jilin isolates (77/89) were resistant to all tested carbapenems, and 75 of them were at least sensitive to one or both of tigecycline and colistin B. But one isolate was resistant to all tested antibiotics including tigecycline and colistin B.The result of antimicrobial susceptibility is shown in Table n = 34), ST208 (n = 14), ST540 (n = 13), ST1779 (n = 6), ST368 (n = 3), ST218 (n = 2) and ST369 (n = 2) as the majority. All newly discovered STs just occupied one isolate in this study. The previously registered ST75 was not found in this study.Among 89 Jilin isolates, 22 STs were identified, among them ten STs including ST2370, ST2393, ST2395, ST2396, ST2397 ST2398, ST2399, ST2400, ST2401, and ST2402 were found for the first time. It should be noted, ST1779, with 6 isolates, was not recognized as novel ST in MLST Database, but the clues to the existence of ST1779 was hardly to be founded. The frequencies of STs ranged from 1–34 with ST195 (n = 3), ST373 (n = 1), ST699 (n = 1), ST2395 (n = 1), ST2396 (n = 1), ST2397 (n = 1), ST2398 (n = 1), ST2399 (n = 1), ST2401 (n = 1), and ST2402 (n = 1) were found to be sensitive to at least one of tested carbapenems, and among them just ST195, ST373 and ST699 were previously discovered STs. The occupation of carbapenems sensitive isolates in all previously discovered STs was about 7.6% (6/79), while that in newly discovered STs was 70% (7/10). The attempts have been done to ensure the association between the STs and the antimicrobial susceptibility. In this study, ST373 (n = 1), ST699 (n = 1), ST2395 (n = 1), ST2396 (n = 1), ST2397 (n = 1), ST2399 (n = 1), ST2401 (n = 1), ST2402 (n = 1) were found to be sensitive to ertapenem, imipenem and meropenem in general, the overwhelming majority isolates of other STs are non-sensitive to three carbapenems antimicrobial, and association was statistically significant . The association between the STs and the carbapenem sensitivity was showed in Fig. ST 195 (n = 6) is not a novel ST, it has never been founded in MLST database before.ST195, ST208, ST218, ST368, ST369, and ST540, which are predominant STs in Jilin could be classified into CC92 group. However, other main STs in Jilin including ST1779, ST1926, and ST2370, could not be classified into CC92 group because of lacking sufficient alleles of the loci in housekeeping genes. It must be pointed out that although ST1779 , ST195 (n = 36), ST92 (n = 32), ST208 (n = 25), ST75 (n = 22), ST540 (n = 19), ST1417 (n = 17), ST138 (n = 10), ST191 (n = 10) and ST368 (n = 8). Jilin predominant STs shared same group founder ST369 with all STs of Guizhou (southeast China), and some sporadic isolates from other regions. But predominant STs in Guizhou were ST1646, ST1417, ST1145, ST2200, ST2208, ST2207, ST2204, ST1658, and ST1144 in Guizhou, which showed different origin and evolution trend from Jilin. The aggregation trend was not found in STs isolated from Zhejiang (east China), Shanghai (east China), Guangdong (south China), Fujian (south China), and Beijing (north China)  was about 0.247 (22/89) was not high, but the proportion of newly discoved ST and their origin were over our expectation. Ten novel STs were identified in 89 isolates, it could be said that the results of this study contributed newer STs to the MLST database. And many of newly discovered STs just with 1 isolate were regarded as singleton lacking relationship with previous discovered Chinese STs, but had closer relationship with foreign STs, which suggested that they are maybe the variant of foreign strains. The new immigrant with strong resistance, such as ST2933, should be paid more attention to continuously monitor and control its epidemiology because it will get more opportunities to spread and are more likely to become dominant strains in the future.A. baumannii in this hospital. As a nation level major ST, ST208 had many variants and evolution branches  of Jilin STs belonged to CC92 group, which is the largest and most widely distributed 1.1% of Jin China –17, epidin China , 19 and in China . And theA. baumannii have increased because of the acquirement of mobile genetic elements such as transposons, plasmids, and integrons. Especially, carbapenems resistant A. baumannii (CR-Ab) is a worldwide problem because these strains are often resistant to all other commonly used antibiotics [Multidrug resistance strains (MDR) of ibiotics . In the ibiotics , we testA. baumannii or A. colcaoaceticus, which makes it impossible to include such strains in our study. Although the results of this study showed the unique molecular epidemiological characteristics of A. baumannii in Jilin Province, a single origin of isolates and a low number of isolates are also the shortcomings of this study. It will be more meaningful to obtain more isolates from more hospitals for similar study in the future.Beside Jilin province, the patients also came from other domestic regions and neighboring countries. All strains were identified by Matrix Assisted Laser Desorption Ionization (MALDI)-Time of Light (TOF)-Mass Spectrometer (MS) and 16SrRNA encoding sequence analysis. However, there are still some strains that cannot be identified as A. baumannii is very serious in Jilin Province, the proportion of bacteria resistant to at least one carbapenems antibiotic has exceeded 94% (84/89). Antibiotic resistance is a helpful factor for predominant STs spreading. The proportion of predominant STs in Jilin are different from some domestic regions, such as Guizhou, Beijing and Hunan, and suspicious nation cross new STs occupied high proportion in Jilin. Epidemiological research by MLST in single hospital is helpful to monitor and control nosocaomial infection of A. baumannii because newly discovered STs, genetic relatedness and geographical distribution reflected sufficient spreading clues of A. baumannii.Overall, the antibiotics resistance of All bacteria are routinely collected for diagnosis in the Affiliated Hospital of Changchun University of Chinese Medicine. No extra samples and personal information about patients were used in the study. Therefore, a written personal informed consent and ethics committee approval were not required, and the study was in line with Chinese laws.n = 89) clinical isolates of A. baumannii were collected from the Affiliated Hospital of Changchun University of Chinese Medicine during 2013–2017. All the isolates were identified by automated VITEK2 and confirmed by 16SrRNA sequencing. For the preparation of DNA, bacteria were cultured overnight in Luria–Bertani (LB) broth at 37℃ in high containment facility, a biosafety level 2 facility at College of Basic Medical Science of Jilin University. Total DNA was extracted by EasyPure® Bacteria Genomic DNA Kit (TransGen Biotech) under the protocol and stored at -20℃ for further use.Eighty-nine  [Escherichia coli ATCC® 25,922 and Pseudomonas aeruginosa ATCC® 2785 were included as controls. Breakpoints of ertapenem , imipenem , meropenem , tigecycline , and colistin B  were the CLSI guidelines clinical breakpoints for Acinetobacter spp. The association between STs and the antimicrobial susceptibility among the study population was carried out using Fisher’s precision probability test. And all antibiotics were the products of Sigma.All collected e (CLSI) . Escherihttps://pubmlst.org/primers-used-mlst-acinetobacter-baumannii-complex-oxford-scheme) were used to amplify gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD, which are conserved housekeeping genes in MLST analysis (Table https://pubmlst.org/primers-used-mlst-acinetobacter-baumannii-complex-oxford-scheme). PCR amplification, sequence analysis and determination of ST for each isolate were carried out according to online A. baumannii typing protocols (https://pubmlst.org/bigsdb?db=pubmlst_abaumannii_seqdef). The PCR amplicons were double pass sequenced using commercial SANGER sequencing services . Novel sequence types (STs) were submitted to the A. baumannii MLST Database.MLST was carried out according to the ‘Oxford’ scheme . Primersis Table . (https:http://eburst.mlst.net/) [The relatedness analysis was performed by online goeBURST and eBURST under the criteria of single locus variant (SLV) as both goeBURST and eBURST allow unrooted tree-based relationship representation of the analyzed isolates ( st.net/) , 25. Thehttp://pubmlst.org/bpseudomallei), which is a flexible tool for relationship and regional characteristic analysis [Nucleotide sequence diversity (π) was calculated using DNAsp V5.1 software . All Chianalysis .To further determine the molecular evolutionary relationship between Chinese STs and prevalent STs in other countries, 22 Jilin STs, another 10 Chinese epidemic STs (isolates ≥ 8), and 120 epidemic STs (isolates ≥ 2) from other countries were selected and analyzed by Unweighted Pair Group Method (UPGM) of PHYLOViZ program , 29. In


To evaluate whether an impaired anterior visual pathway  is associated with underlying beta-amyloid (Aβ) pathologies in patients with Alzheimer’s disease dementia (ADD) and mild cognitive impairment (MCI), we compared retinal structural and vascular factors in each subgroup with positive or negative amyloid biomarkers. Twenty-seven patients with dementia, thirty-five with MCI, and nine with cognitively unimpaired (CU) controls were consecutively recruited. All participants were divided into positive Aβ (A+) or negative Aβ (A−) pathology based on amyloid positron emission tomography or cerebrospinal fluid Aβ. The retinal circumpapillary retinal nerve fiber layer thickness (cpRNFLT), macular ganglion cell/inner plexiform layer thickness (mGC/IPLT), and microcirculation of the macular superficial capillary plexus were measured using optical coherence tomography (OCT) and OCT angiography. One eye of each participant was included in the analysis. Retinal structural and vascular factors significantly decreased in the following order: dementia < MCI < CU controls. The A+ group had significantly lower microcirculation in the para- and peri-foveal temporal regions than did the A−. However, the structural and vascular parameters did not differ between the A+ and A− with dementia. The cpRNFLT was unexpectedly greater in the A+ than in the A− with MCI. mGC/IPLT was lower in the A+ CU than in the A− CU. Our findings suggest that retinal structural changes may occur in the preclinical and early stages of dementia but are not highly specific to AD pathophysiology. In contrast, decreased temporal macula microcirculation may be used as a biomarker for the underlying Aβ pathology. Alzheimer’s disease dementia (ADD) is the most common neurodegenerative dementia in the elderly and is associated with the accumulation of beta-amyloid (Aβ) plaques and tau neurofibrillary tangles . DiagnosPatients with ADD commonly show visuospatial dysfunction that is mainly attributed to damage of the parieto-occipital or temporo-occipital visual pathways . Ocular Cerebrovascular dysfunction in association with vascular deposition of Aβ is one of the major pathophysiological mechanisms of AD . The braSeveral studies using laser Doppler ultrasonography or a retinal function imager (RFI) have revealed narrowed central retinal vessels with a decreased blood flow rate in patients with mild cognitive impairment (MCI) and ADD ,12,13. AOn the other hand, OCT angiography (OCTA) can be used to non-invasively visualize the retinal microvascular network involving multiple retinal layers in a reproducible manner without a contrast agent . Prior sIn this study, we first analyzed the structural changes in the mGC/IPLT and cpRNFLT with microvasculature in patients with clinically diagnosed dementia, MCI, and in cognitively unimpaired (CU) controls using OCT and OCTA, and we investigated the diagnostic performance  of OCT/OCTA variables discriminating dementia and/or MCI group from the CU group. Second, to evaluate whether the impaired anterior visual pathway  is truly associated with the underlying amyloid pathology, we further compared the OCT and OCTA parameters in each subgroup with positive or negative amyloid biomarkers and examined their usefulness as biomarkers for predicting the underlying amyloid pathology.We recruited 234 eyes of 117 participants with clinically diagnosed dementia, MCI, or CU at the Neurological Clinic of Pusan National University Hospital from September 2019 to July 2021. All participants were evaluated by a neurologist (E.J.K.) with expertise in neurodegenerative diseases who underwent clinical interviews, neurological examinations, and detailed neuropsychological testing . PatientThorough ophthalmologic examinations, including best-corrected visual acuity (BCVA), slit lamp examination, intraocular pressure (IOP) measurement using Goldmann applanation tonometry, gonioscopy, dilated fundus examination, stereoscopic optic disc, and RNFL photography , biometry using IOLMaster , standard automated perimetry , ultrasonic pachymetry , and keratometry  were conducted in all participants.The exclusion criteria were glaucoma, macular degeneration, retinal vascular disease, including diabetic retinopathy and retinal vein occlusion, corneal or ocular media opacity, history of ocular trauma, astigmatism ≥ 3.0 diopter, spherical equivalent ≥ 6.0 diopter, BCVA < 20/40, and any ocular surgery except for uncomplicated cataract extraction. Additional medical exclusion criteria included uncontrolled hypertension that caused vascular eye changes .ClinicalTrials.gov . Written informed consent was obtained from all participants enrolled in the study.This study was approved by the institutional review board of the Pusan National University Hospital  and registered at 2) and the optic disc cube 200 × 200 scan protocol (6 × 6 mm2) were performed for each eye. The software algorithm automatically identified the outer boundaries of the RNFL and IPL. The mGC/IPLT, within a 14.13 mm2 elliptical annulus area centered on the fovea, was calculated as the distance between these two boundaries. The average, minimum, and six sectoral  mGC/IPLT values were measured -OCT 5000 with AngioPlex™ , at a scan rate of 68,000 A-scans per second and a central wavelength of 840 nm, was used to measure the mGC/IPLT and cpRNFLT. After pupil dilatation, the macular cube 200 × 200 scan protocol  of the FAZ were measured. A circularity index closer to 0 indicates an irregular shape, and a value closer to 1 indicates a circular shape [ZEISS Angioplex™ OCT angiography has tracking software known as FastTracrtifacts . The OCTrtifacts . In macurtifacts . The SCPrtifacts . Three crtifacts ,31. VD war shape .All OCT and OCTA scans were performed by an experienced examiner, and all scans were reviewed individually by two investigators (S.M. and J.L.) for quality evaluation (segmentation errors and motion artifacts). Substandard scans or image scans with a signal strength of <7 were excluded.Data distribution normality was checked using the Kolmogorov–Smirnov test. One-way analysis of variance (one-way ANOVA) and the Kruskal–Wallis test with post-hoc correction for multiple comparisons were used to analyze differences in demographic and ophthalmological characteristics in continuous variables among the clinical diagnostic groups. To analyze differences in categorical variables, we used the chi-square test or Fisher’s exact test. The Student’s t-test or Mann–Whitney U test for continuous variables was used for comparison between the A+ and A− groups after adjustment for the Mini-Mental Status Examination (MMSE). The correlation between OCT or OCTA parameters and MMSE scores was estimated using Pearson or Spearman’s rank correlation coefficients.Receiver operating characteristic (ROC) curves were drawn by plotting sensitivity against 1-specificity, and areas under the ROC curves (AUCs) were used to evaluate the diagnostic performance of the OCT/OCTA parameters. Ideal cut-off points were derived using the Youden index . Binary http://cran.r-project.org accessed on from September 2021 to December 2022), version 4.0.5, and SPSS software, version 22.0 . In all analyses, a p-value < 0.05 was considered statistically significant.All statistical analyses were performed using the language R (n = 69), CSF studies for Aβ analysis (n = 11), or both (n = 9).Among the 234 eyes of the 117 participants, 120 eyes were excluded during screening . One eye of each participant was included in the analysis. In cases where both eyes met the eligible criteria, the eye with the worse mean values of structural and vascular parameters on OCT/OCTA images was selected [n = 42 eyes) or A− (n = 29 eyes) groups. The clinical diagnostic groups were classified as A+ or A− as follows: 22 eyes (A+) and 5 eyes (A−) of patients with dementia, 17 eyes (A+) and 18 eyes (A−) of patients with MCI, and 3 eyes (A+) and 6 eyes (A−) of CU controls. The mean interval between the OCT/OCTA test and the amyloid PET scan or CSF study for Aβ1-42 was 1.1 ± 1.0 years. The proportions of eyes (number) from A+ participants in the dementia, MCI, and CU control groups were 81.5%, 48.6%, and 33.3%, respectively. These differences differed significantly among the clinical groups (p = 0.008).Based on amyloid PET or CSF Aβ level results, all participants were subdivided into A+ , 9 clock-hour cpRNFLT , and perifoveal temporal VD  parameters were more accurate than the others in discriminating underlying Aβ from non-Aβ pathology, with cut-off points of 0.41, 54.0 μm, and 16.88 mm/mm2, respectively. In the CU group, the structural factors, superotemporal, inferior, and inferotemporal mGC/IPLT, vascular factors, FAZ circularity index, and para- and peri-foveal temporal VD and PD showed accurate diagnostic performance in differentiating the A+ from A− groups (0.775 ≤ AUCs ≤ 0.988) , PD , and parafoveal temporal PD  were more accurate than other parameters in differentiating the A+ from A− groups, with cut-off points of 16.88 mm/mm≤ 0.988) . The par≤ 0.988) .2; PD, 36.3%) than in the A− MCI participant .p = 0.018) was significantly correlated with MMSE scores among all participants.The FAZ circularity index , 3 clock-hour cpRNFLT ) and vascular ) parameters were positively correlated with MMSE scores in the entire A− group, no significant structural or vascular parameters were correlated with MMSE scores in the entire A+ group.Whereas structural  Among the clinical diagnostic groups, as the severity of neurodegeneration increased, the structural and vascular parameters of OCT/OCTA tended to significantly decrease. The superior quadrant cpRNFLT  and the FAZ circularity index (as a vascular factor) showed the best performance in differentiating dementia and/or MCI from CU controls. (2) The A+ group had significantly decreased microcirculation in the para- and peri-foveal temporal regions compared to those of the A− group. (3) In the dementia group, there were no significant differences in the structural or vascular parameters on OCT/OCTA according to Aβ positivity. However, in the MCI group, the A+ group had a greater temporal quadrant and 9 clock-hour cpRNFLT and lower para- and peri-foveal temporal VD and PD than those of the A− group. (4) In the CU group, the average, temporal, and inferior mGC/IPLT and FAZ circularity decreased more in the A+ group than in the A− group. (5) FAZ circularity was correlated with MMSE scores in all participants. However, no such correlation was observed in the A+ group.The results of the present study, which showed a reduced superior quadrant and inferior cpRNFLT region in patients with dementia and/or MCI compared to those of CU controls, partially correspond to those of previous studies conducted in patients with ADD ,6,35,36.Regarding the retinal microvasculature, in the present study, the FAZ circularity index and perifoveal temporal and mean vascular parameters (VDs and PDs) were reduced in patients with dementia or MCI compared to those of CU controls. This is also partly consistent with the results of previous studies that reported FAZ enlargement or reduced macular microcirculation in patients with clinically diagnosed ADD compared to controls ,18,19,37Several recent studies conducted on patients with or without Aβ pathology have shown inconsistent results ,22,23,40In our study, although significantly reduced vascular parameters were detected in the whole A+ versus A− group, no differences in structural parameters were found. In the dementia group, no differences in OCT/OCTA parameters were observed between the A+ and A− groups. In contrast, the MCI and CU groups had significantly different parameters between the A+ and A− groups. Each A+ group may have underlying Aβ pathology, but the underlying pathology in each A− group may be speculated differently. While the A− dementia group would have various underlying pathologies other than Aβ, A− MCI or A− CU participants seemed to have a very low risk of progression to neurodegenerative disease . TherefoThe unexpectedly increased nine clock-hour and temporal cpRNFLT in the A+ MCI group may be explained by a paradoxical increase in RNFLT in the MCI stage, attributed to gliosis occurring prior to neuronal loss . ApproxiIn contrast to the heterogeneous structural changes in the retina, a decrease in the para- or peri-foveal temporal microcirculation was consistently observed in the A+ group. Magnocellular neurons (M cells), the largest class of RGCs, are known to be primarily affected in AD, and the number of M cells is greatest in the temporal macula ,48. Thern = 6).Lastly, we detected a reduction in the FAZ circularity index and peri/parafoveal temporal VD and PD between the A+ dementia and A− CU groups, which was incompatible with the results of Hann et al.’s study ; howeverA major limitation of our study is the small sample size in each clinical group. No significant differences of OCT and OCTA parameters among clinical groups or between A + and A− groups were observed after correction for multiple testing, which might be due to the low statistical power caused by multiple variables considering the small and imbalanced sample sizes between the groups. For such a reason, we also acknowledge that the OCT and OCTA parameters with the best discriminating performances from the ROC curve were no longer significant in the binary logistic regression model after correction for multiple testing.This is the first study to investigate the role of Aβ pathology in each clinical diagnostic group: dementia, MCI, and CU controls. Significant reductions in cpRNFLT and the FAZ circularity index were found to be associated with neurodegenerative processes, but not with or without Aβ pathology. Therefore, loss of the superior quadrant cpRNFLT and a decreased FAZ circularity index may be considered the most consistent phenomena in various neurodegenerative diseases, and are not highly specific to AD. In contrast, decreased para- and peri-foveal temporal microcirculation might be a good biomarker for predicting the underlying Aβ pathology. Longitudinal studies with larger samples, well-defined by biomarkers, are needed to evaluate retinal structural and neurovascular changes associated with disease progression in various neurodegenerative diseases.


Peripheral nerve injury (PNI) is a neurological disorder that causes more than 9 million patients to suffer from dysfunction of moving and sensing. Using biodegradable polymers to fabricate an artificial nerve conduit that replicates the environment of the extracellular matrix and guides neuron regeneration through the damaged sites has been researched for decades and has led to promising but primarily pre-clinical outcomes. However, few peripheral nerve conduits (PNCs) have been constructed from controllable biodegradable polymeric materials that can maintain their structural integrity or completely degrade during and after nerve regeneration respectively. In this work, a novel PNC candidate material was developed via the electrospinning of polyhydroxy butyrate/chitosan (PHB/CS) composite polymers. An SEM characterisation revealed the resultant PHB/CS nanofibres with 0, 1 and 2 wt/v% CS had less and smaller beads than the nanofibres at 3 wt/v% CS. The water contact angle (WCA) measurement demonstrated that the wettability of PHB/CS electrospun fibres was significantly improved by additional CS. Furthermore, both the thermogravimetric analysis (TGA) and differentiation scanning calorimetry (DSC) results showed that PHB/CS polymers can be blended in a single phase with a trifluoracetic solvent in all compositions. Besides, the reduction in the degradation temperature (from 286.9 to 229.9 °C) and crystallinity (from 81.0% to 52.1%) with increasing contents of CS were further proven. Moreover, we found that the degradability of the PHB/CS nanofibres subjected to different pH values rated in the order of acidic > alkaline > phosphate buffer solution (PBS). Based on these findings, it can be concluded that PHB/CS electrospun fibres with variable blending ratios may be used for designing PNCs with controlled biodegradability.  Electrospinning is a versatile technique that can efficiently fabricate micro and nanosized polymer fibres. A nanofibrous scaffold can effectively mimic the microstructure in the extracellular matrix based on its structural properties. Electrospun fibres have unique microstructure, including a high surface-to-volume ratio and porosity, therefore, can be used in electronic devices, filters, tissue implants, drug delivery and other biomaterial applications ,2,3. ElePolyhydroxybutyrate (PHB) has been reported as a novel biodegradable polymer that is used in biomedical applications, such as biocontrol agents, drug carriers, tissue implants, anti-osteoporosis agents, etc. ,5,6. PHBChitosan (CS) has gradually attracted enormous interest in tissue engineering and regeneration over the last few years because it shows several advantages, including biocompatibility, biodegradability, hydrophilicity, nontoxicity and anti-microbial activity with cells . HoweverBoth PHB and PHB/CS electrospun fibres have been reported to be used as nerve conduits with growth factors that successfully guided neurite outgrowth after two months of implantation ,9. HowevMany studies have been carried out on either the hydrolytic or enzymatic degradation behaviours of biodegradable polymers such as polylactic acid (PLA), polycaprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) and others ,15,16. FIn previous studies, a PHB/CS blend solution was optimized and fabricated by electrospinning into nanofibres for tissue engineering applications ,19,20,21Materials®, 99%, Merck), chitosan (CS) .Polyhydroxybutyrate (PHB) , trifluoroacetic acid (TFA) , neutral (pH = 7) and alkaline solutions (pH = 12)2) and pre-dried in a vacuum oven for 24 h before the experiments. The scaffolds were then submerged in 10 mL of the acidic, PBS and alkaline solutions in individual tubes with the screw caps tightened and maintained at 37 °C for 8 weeks. All solutions were changed every week. For each data point, three replicates were prepared to minimize the effect of random errors. The scaffolds were removed at specified intervals for analysis, at which time they were rinsed thoroughly with de-ionized water, dried and placed in an oven at 35 °C for 24 h until a constant weight was obtained.The degradation properties of the electrospun PHB/CS nanofibre samples were analysed in three different solutions: (1) The acidic solution was prepared using acidic lactate sodium (ALS) and adjusting the pH to 2 by adding HCl. (2) The neutral solution was prepared using PBS at pH 7.4. (3) The alkaline solution was prepared by adjusting the pH value of PBS to 10 by adding NaOH. Samples were cut into squares (1 × 1 mmScanning electron microscope (SEM)Different microfibres were analysed using a Hitachi 8230 field emission scanning electron microscope  after coating the samples with Au in a sputter. The diameters of the fibres and beads were measured from the SEM images using ImageJ software. Fifty measurements were carried out for each sample.Fourier transform infrared spectroscopy (FTIR)−1 to 400 cm−1 wavenumbers with a 2.0 cm−1 resolution.FTIR spectroscopy was performed to investigate the functional groups present in the electrospun PHB/CS nanofibres. Before the test, PHB/CS electrospun fibres were rinsed with de-ionised water and dried in a vacuum dryer until all the water was removed. The FTIR spectra of the PHB/CS electrospun fibres were obtained from a 5 min scan ranging from 4000 cmContact angle measurementsThe wettability of the electrospun fibres mats was evaluated through the sensile drop water contact angle test using an OCA 15 plus contact angle measurement system  equipped with a CCD camera (precision ± 0.2°). All the measurements were performed by applying ultra-pure water at room temperature and 10–15% humidity. The average values were evaluated based on five random points of each specimen.Thermalgravimetric analysis (TGA)The samples were subjected to TGA in a PerkinElmer 2000 instrument under a continuous nitrogen flow of 40 mL/min. Approximately 10 mg samples were prepared and then heated in a ceramic crucible from 50–400 °C at the heating rate of 10 °C/min. Differential scanning calorimetry (DSC)DSC was performed by adding an approximately 2 mg sample to an aluminium crucible. The sample was heated from room temperature (25 °C) to 550 °C at the heating rate of 10 °C/min. Nitrogen was used with a gas flow rate of 20 mL/min. All samples were tested under the same conditions to obtain comparable TGA curves.Statistical analysisp-value of less than 0.05 was considered as statistically different.The significance differences of the degradation results in acidic, neutral and alkaline solutions from PHB/CS electrospun fibres were analysed using a one-way ANOVA test in Prism 8. A All PHB/CS scaffolds were formed in one single phase and prepared by electrospinning techniques. −1, which was assigned to the stretched vibration of the C=O group. Furthermore, the crystallinity phase of PHB in the FTIR spectrum was shown with the peaks observed at 816–826 cm−1, 1276–1278 cm−1 and 1720–1722 cm−1. The peaks of the amorphous phase arose at 1130 cm−1. Interestingly, the symmetric wagging of the CH3 group in PHB showed at 1378 cm−1. For CS, the characteristic peak was at 3300–3550 cm−1, representing the stretching OH and NH groups. The amide I that presented in CS was at 1540 cm−1. From −1 to shift to a lower wavenumber. Moreover, the peaks at 973 cm−1 and 1293 cm−1 shifted to higher wavenumbers. After acidic and alkaline degradation, all samples showed a broader peak at 3550 cm−1, and a strong stretching C=O signal peak formed at 1750 cm−1. This is because the crystalline phase of PHB was disturbed by addition CS, which led to rise in the peak of the amorphous phases. The presented hydrogen bond dominated the increase in the amorphous phases.To evaluate the surface hydrophilicity of the PHB/CS nanofibrous mats with different CS contents, the water contact angles (WCA) on the PHB/were measured consecutively as shown in The TGA curves of all PHB/CS fibrous mats were compared with pure PHB and CS to analyse their variation in degradation temperature. The TGA and DTA curves are shown in p < 0.0001) and PHBCS2 (p < 0.0005), but no critical difference was detected in PHBCS1 (p > 0.01). In the alkaline conditions, the remaining mass percentages at week 12 in each sample were 71.33 ± 1.14 (PHBCS0), 19.90 ± 2.3 (PHBCS1), 13.28 ± 5.48 (PHBCS2) and 0 (PHBCS3). The results showed significant differences in the remaining mass in all other samples compared to PHBCS0 (p < 0.0001).The in vitro characterisation of degradation with various pH levels illustrated in −1 appeared in alkaline solution after 4 weeks of degradation. This is because NaOH can activate the hydroxide groups on PHB/CS fibres’ surfaces [−1 was assigned to the absorption of helical (α type) crystals, the 979 cm−1 peak was also assigned to the crystalline phase at C-C stretching, and the peak at 1180 cm−1 was assigned to the amorphous phase. In our results, the peaks at 973 cm−1 and 1293 cm−1 shifted to higher wavenumbers because the crystalline phase of PHB was disturbed by the addition of CS, which led to the peak of the amorphous phases increasing. The presented intra-molecular hydrogen bond dominated decrease in the amorphous phases [In FTIR, an O-H stretching bond at 3550 cmsurfaces ,23. Mores phases .m in the DSC curves. Furthermore, the reduction in crystallinity from PHBCS0 to PHBCS3 can be explained by the fact that the rigid CS molecules disrupted the lamellar thickness of the PHB crystallites and suppressed PHB crystallisation [In lisation ,26.In the WCA analysis, the increased CS contents demonstrated a higher degree of hydrophilicity, which is based on the increasing number of terminal hydroxyl and amino groups of the CS chains, which can easily interact with water molecules via H-bonding.For the in vitro degradation, PHB/CS showed different behaviours with different CS contents under different pH levels. With the increased CS contents, both PHB and CS indicated an increase in the degradation rate at different pH levels. This may be explained that the loss of CS increased the water diffusion possibility, which increased the contact areas between PHB and water. Moreover, a reduction in crystallinity and an increase in hydrophilicity can accelerate the hydrolysis in PHB/CS scaffolds. Compared to PBS (pH = 7) solution, PHB/CS degraded much faster in both sodium lactate/HCl (pH = 2) and NaOH solutions (pH = 10). Interestingly, PHBCS3 completely broke into pieces after 12 weeks of degradation in both acidic and alkaline solutions. This is because the reduction in the crystallinity and hydrophobic nature of PHBCS3 which resulted in faster water penetration through the structure. Besides, in acidic conditions, samples with additional CS experienced a much faster degradation rate than those without CS. This is due to the weak base nature of CS ,28 that This research has discovered the physical, chemical and microstructure behaviours of PHB/CS electrospun fibres under acidic, neutral, and alkaline conditions with different CS contents, which provides a view of the nano-surface morphology change of PHB/CS. We found that the PHB/CS electrospun mats were successfully fabricated by the electrospinning process as a single phase in each composition. Moreover, with increasing additions of CS, PHB/CS electrospun fibres have reduction in both hydrophobic nature and crystallinity. Furthermore, we analysed the degradability of PHB/CS in acidic, neutral, and alkaline conditions. The overall trends showed that the degradation rate increased with the addition of CS at all pH values. Furthermore, the degradability of PHB/CS between different pH values were ranked from acidic > alkaline > phosphate buffer solution (PBS). Both acidic and alkaline environments will accelerate PHB/CS degradation, and completely dissolve in pieces of at week 12. Comparatively, acidic environments can perform a more violent degradation of the PHBCS scaffold than in alkaline and PBS solutions.


Cancer is a major disease burden to society. Increasing evidence has indicated that low skeletal muscle mass is linked with cancer prognosis. The purpose of the study is to determine the impact of preoperative low skeletal muscle mass (LSMM) on complications and survival of patients who undergo laparoscopic gastrectomy for gastric cancer (GC).This study retrospectively collected patients undergoing laparoscopic gastrectomy for GC between January 2017 and December 2018. Tumor staging was performed according to the American Joint Committee on Cancer 8th edition. The third lumbar psoas index (PMI) was assessed by computed tomography (CT) within 15 days before surgery. Postoperative complications were classified according to Clavien-Dindo classification and dichotomized into none vs any . Using propensity score matching (1:1) to obtain 2 well-balanced cohorts for available variables influencing clinical outcomes, comparing the postoperative complications and 3-year overall survival (OS) between LSMM group and non-LSMM group.n = 292) were men and 24.35% (n = 94) were women. A total of 249 (64.51%) patients were diagnosed with LSMM. Compared with the non-LSMM group, the LSMM group manifested significantly shorter 3-year OS . However, the incidence of postoperative complications was no difference between two groups after matching. After stratification based on the pT stage of the tumor, statistically significant difference in the 3-year OS rates of the advance GC cohort between the two groups were observed.A total of 386 patients, 226 were matched for analyses. The average patient age was 57.31 ± 10.33 years; 75.65% (LSMM predicts a poor prognosis for patients with advance GC and it is not associated with postoperative complications. Gastric cancer (GC) is one of the most common gastrointestinal malignancies worldwide. According to the latest global cancer statistics, GC is the 5th most common malignant tumor and the 4th leading cause of cancer-related mortality –3. With Skeletal muscle depletion initially defined as the progressive loss of skeletal muscle mass as a result of the aging process . RecentlPropensity score analysis is a statistical technique commonly used to evaluate the efficacy of clinical interventions in observational studies , 17. ProIn this study, we focused on skeletal muscle mass by measuring bilateral psoas muscles mass using computed tomography (CT) scan before surgery. The primary aim of the study was to ascertain the effect of preoperative low skeletal muscle mass (LSMM) on postoperative complications and OS rate via using PSM.Patients who underwent laparoscopic gastrectomy for primary GC at our hospital between January 2017 and December 2018 were enrolled in this retrospective study. The inclusion criteria: (1) patients who were pathologically diagnosed as primary GC; (2) patients who were administrated for the primary diagnosis and were therapy naive; (3) patients who were performed laparoscopic gastrectomy. Patients were excluded if (1) they had chemoradiotherapy, targeted therapy before surgery; (2) they had identified metastasis before surgery; (3) CT examination was not performed within 15 days before surgery; (4) their clinical data and follow-up data were incomplete or non-detailed. The study was approved by the Ethics Committee of Shandong Provincial Hospital.All relevant data were retrospectively collected from the hospital database, including age, sex, body mass index (BMI), nutrition risk screening 2002(NRS2002), tumor grade, TNM stage, albumin, total protein, hemoglobin, lymphocyte percentage (LYM%), platelets, white blood cell (WBC), type of resection, comorbidities and 1-, 3-year follow-up records.2/m2 or less for men and 3.92 cm2/m2 or less for women  images of the L3 level on two consecutive transverse sections was used to assess psoas muscle mass. To calculate the PMI, bilateral psoas muscles mass assessed by CT scan is divided by the square of the patient’s height. LSMM was accepted when the PMI was 6.36 cmulations ). All CTAccording to the international classification of gastric cancer, early gastric cancer is defined as a lesion confined to the mucosa or submucosa and presence or absence of regional lymph node metastasis, while advanced gastric cancer is defined as T2–T4 cancer without distant metastasis. TNM staging was based on the American Joint Committee on Cancer (AJCC) 8th edition. The surgical methods of gastrectomy include total gastrectomy and subtotal gastrectomy. Subtotal gastrectomy includes proximal gastrectomy and distal gastrectomy. In the study, postoperative complications were defined as surgical related adverse events within 30 days after surgery. Postoperative complications were classified according to Clavien-Dindo classification and dichotomized into none vs any .t-test or Mann-Whitney U-test. Categorical variables were analyzed by χ2 test or Fisher’s exact test. Patients in the cohorts of LSMM and non-LSMM were propensity score-matched at a 1:1 ratio using the Nearest Neighbor Matching approach with a caliper = 0.2. Propensity Score Matching based on age, preoperative BMI, albumin, NRS2002, LYM%, WBC. A univariate and a multivariate sensitivity analysis were also performed. Kaplan–Meier method and Log-rank test were performed to conduct survival analyses and evaluate differences in survival time, respectively. Values of p < 0.05 were considered statistically significant. The assessment of propensity score matching is also shown . Continuous variables were compared between groups using the independent so shown .n = 292) were men and 24.35% (n = 94) were women. A total of 249 (64.51%) patients were diagnosed with LSMM , BMI (p < 0.0001), NRS2002 (p < 0.0001), albumin (p < 0.0001), LYM% (p = 0.01) and WBC (p = 0.006) between the two groups, but no significant differences were observed in the sex, tumor histological grade, pT Stage, pTNM Stage, comorbidities or type of resection    . After triables) .p = 0.036), hemoglobin (p = 0.03), smoker (p = 0.014). There were no statistically significant differences in the complications between the LSMM group and non-LSMM group after propensity score matching (p = 0.654) (The distribution of postoperative complications is listed in = 0.654) .p = 0.016, The median follow-up time of the entire matched cohort was 36 months (range 0–45 months). There are 65 deaths. The 3-year OS rates was 71.24% for all patients. The 1-, 3-year OS rates were 83.2%, 64.6% respectively, for patients with LSMM and 92.9%, 77.9%, respectively, for those non-LSMM. The survival curves for patients of GC with and without LSMM are shown in p = 0.286, p = 0.034, After stratification based on the pT stage of the tumor, the 3-year OS rate of the early GC cohort was 89.66%, and no statistically significant difference in the 3-year OS rates between the LSMM  and non-LSMM  were found (p = 0.016) and that LSMM was an independent risk factor for overall survival in patients with GC. Furthermore, preoperative LSMM may be particularly useful in advance GC. However, the results in our study showed that LSMM is not associated with the incidence of postoperative complications.The study demonstrated that the 3-year OS rate of patients with LSMM was 64.6%, which was significantly poorer than those with non-LSMM (77.9%) , but no difference was found in early GC . The initial hypothesis of the study was that the two groups can show differences in early and advanced GC. This is an interesting discovery, and no relevant research has been reported so far. We will continue to follow up these patients to further observe the impact of LSMM on the prognosis of patients with early GC.In this study, we divided patients into early stage and advanced stage GC for analysis. The results showed that there was significant difference in 3-year OS between the LSMM group and the non-LSMM group in advanced GC (CT is a routine examination for patients with GC before surgery, so it is feasible to use CT to assess skeletal muscle mass. It will not bring additional economic burden to patients. This study is a single-center retrospective study. The main purpose is to clarify the relationship between preoperative skeletal muscle mass and prognosis in patients with GC. CT was used to assess the preoperative skeletal muscle mass and found high-risk patients. This is of great significance to the scientific diagnosis and treatment of GC patients.There are some potential limitations in this study. This study is a retrospective single-center study, the data integrity may be insufficient. We plan to conduct a prospective study to further explore the adverse effects of LSMM.By using PSM analysis to balance the differences between cofounding variables, the study has proved that LSMM is an unfavorable factor for OS rates, especially in patients with advance GC. However, LSMM is not associated with postoperative complications.


Liver-related comorbidities can impair the health-related quality of life (HRQL) in people living with human immunodeficiency virus (HIV) (PLWH). However, the role of hepatic steatosis and significant fibrosis in PLWH remains incompletely characterized. Therefore, the aim of this study was to explore the association of hepatic steatosis and significant fibrosis on the HRQL using the medical outcomes study HIV health survey (MOS-HIV) in PLWH.A total of 222 PLWH were included in the final analysis of this cohort study. Metabolic comorbidities, socioeconomic factors, and HIV-related parameters were assessed. Hepatic steatosis and fibrosis were measured using vibration-controlled transient elastography (VCTE). The MOS-HIV survey, containing two summary scores  and mental health summary (MHS)) and ten domains, was used to assess the HRQL. Clinical predictors were identified using multivariable linear regression models.n = 81, 36.5%). Significant fibrosis was present in 7.7% (n = 17). The mean PHS and MHS scores were 52.7 ± 9.5 and 51.4 ± 10.5, respectively. The lowest scores were in the general health perception (GHP) and energy/fatigue (EF) domains. A high BMI and waist circumference were associated with a poor PHS score. Lower education, unemployment, arterial hypertension, and significant fibrosis remained independent predictors of an impaired HRQL.The majority of this cohort was male, and the median age was 52 years, with a high prevalence of hepatic steatosis (Metabolic comorbidities, significant fibrosis, and a lower socioeconomic status may negatively affect the HRQL in PLWH. Considering the negative impact of significant fibrosis on the outcome, counseling and preventive measures according to current guidelines are recommended in this subgroup of PLWH.The online version contains supplementary material available at 10.1007/s11136-022-03232-w. The life expectancy of people living with human immunodeficiency virus (HIV) (PLWH) has gradually been increasing over the last several decades as a result of advances in antiretroviral therapy (ART) . HIV is Metabolic syndrome and its risk factors have been increasing and show a higher prevalence in PLWH compared to the general population . TreatmeThe HRQL can be assessed with either generic  or disease-specific questionnaires , 21. TheCurrently, only little data is available on hepatic steatosis and fibrosis and their role in affecting the HRQL in PLWH. Therefore, the aim of this study was to explore the association of hepatic steatosis and fibrosis on the HRQL in PLWH using the MOS-HIV survey.n = 8), (b) an incomplete MOS-HIV questionnaire (n = 67) and (c) multiple other reasons (n = 5). A total of 222 PLWH meeting the inclusion criteria were used for this analysis  were approached and assessed by VCTE and the MOS-HIV survey between 2018 and 2021 in this cohort study  at the outpatient clinic of the Metabolic Liver Research Program at the University Medical Center Mainz in Germany. 80 PLWH were excluded from the final analysis due to; (a) invalid VCTE measurements (n = 201 (90.5%), whereas the XL probe was used in n = 21 (9.5%) exams whenever suggested by the device. A total of eight participants were excluded due to invalid measurements. Significant fibrosis (≥ F2) was defined as an LSM ≥ 8.2 kPa ). Alcohol intake was clinically assessed, and consumption above 20 g/day and 10 g/day for males and females was considered a higher alcohol intake. HIV RNA viral load was divided into two groups: below and above the threshold (50 copies/ml).Several characteristics, including sociodemographic variables, metabolic comorbidities, laboratory values, and HIV-related parameters, have been analyzed. Higher education was defined if participants had at least a high school degree, whereas a lower education implies that participants had no high school degree or above. The metabolic syndrome was defined according to the International Diabetes Federation (IDF) . The defFor the assessment of the HRQL, the validated German version of the HIV-specific medical outcomes study HIV health survey (MOS-HIV) was used , 32. TheAll participants provided written informed consent. The study was conducted according to the ethical guidelines of the 1975 Declaration of Helsinki . The ethics committee approved the study protocol of the Landesärztekammer Rhineland-Palatine (Nr. 873.199.10 (7208)).p < 0.05. Univariable analysis was used to examine associations between demographic and clinical variables with the PHS and MHS of the MOS-HIV. All variables with p < 0.05 in the univariable analysis and the two clinical parameters age and sex were then analyzed in a multivariable linear regression model. IBM SPSS Statistic Version 23.0  was used for all data analysis and statistical tests. GraphPad Prism 5.0  was used for all graphs.Quantitative data are presented as median values with interquartile ranges IQR 25th; 75th) or mean values with standard deviation (± SD). Categorical variables are shown as frequencies with percentages. The Mann–Whitney U rank test was used to compare differences between groups of continuous variables  and a lower education was seen in 64.9% (n = 144). The median CAP was 245.5 dB/m . A total of 81 (36.5%) PLWH had hepatic steatosis, of whom 70 (31.5%) participants fulfilled the criteria of a NAFLD. The median LSM was 4.6 kPa , and significant fibrosis was present in 7.7% (n = 17). Arterial hypertension was present in 28.4%. The median BMI and waist circumference were 25.2 kg/m2 and 97.0 cm, respectively. CDC stage C was present in 20.3% (n = 45) of PLWH. Most PLWH were treated with TAF-containing regimens  and 59.9% (n = 133) also received an INSTI as part of the ART regimen. These and other general characteristics of the cohort are summarized in Table A total of 222 PLWH were included for the final analysis in this study. The median age was 52 years , and the median time since diagnosis was 12 years . The majority of PLWH were male sex (70.7%). Unemployment was noticed in 12.2%  and mental health summary (MHS) scores were 56.1  and 53.7 , respectively. The highest scores were seen in the domains physical functioning (PF), role functioning (RF), social functioning (SF), and pain (P). In turn, the lowest scores were seen in the domains of general health perception (GHP), energy/fatigue (EF), and quality of life (QL). The median and mean values of the MOS-HIV are summarized in Table In the univariable analysis, a longer disease duration (time since diagnosis) , female sex , a lower education , unemployment , a diagnosis of sleep apnea syndrome , a high BMI , a high waist circumference , arterial hypertension  and significant fibrosis  were associated with an impaired PHS. In the multivariable analysis, a lower education , arterial hypertension  and significant fibrosis  remained the only independent predictors of a poor PHS , unemployment , arterial hypertension  and significant fibrosis  were associated with an impaired MHS in the univariable analysis. In the multivariable analysis, unemployment , arterial hypertension  and significant fibrosis  were independent predictors of a poor MHS  and mental health (MHS) based on ten domain scores, each evaluating various aspects of someone’s HRQL. In this context, general health perception (GHP) and energy/fatigue (EF) were two of the most burdensome HRQL aspects in this cohort. Arterial hypertension remained an independent predictor of impaired PHS and MHS scores. Lower education and unemployment were independently associated with poor PHS and MHS scores, respectively. To assess the effect of liver disease on the HRQL in PLWH, we included hepatic steatosis and significant fibrosis, measured non-invasively by VCTE, in our analysis. Significant fibrosis remained an independent predictor of a poor HRQL in both summary scores , and the GHP domain was the most burdensome in this subgroup. Overall, the lowest PHS and MHS scores were seen in individuals with significant fibrosis.2) and a high waist circumference may result from poor dietary habits and a lack of physical activity. A lower physical health impacts mental health since the lack of physical activity often aggravates mental well-being in PLWH [Despite the impact of hepatic steatosis and fibrosis on the HRQL in HIV-negative individuals, the association in PLWH remains largely unknown. Hepatic steatosis can be an initiating event for more severe liver injury due to inflammation (steatohepatitis) that promotes scarring of liver tissue (fibrosis). Significant fibrosis can lead to severe liver-related complications and higher mortality . In pati in PLWH . However in PLWH . Sociode in PLWH . A recen in PLWH . Here wePrevious studies have often highlighted a higher prevalence of mental health problems, including anxiety and depression, in PLWH , 38. In The energy/fatigue domain showed one of the lowest scores in this study. Fatigue is a common symptom, especially in patients with chronic liver diseases , 16. In The median values of the two summary scores, PHS and MHS, in this study were comparable to an analysis from Ireland, although the median value of MHS was slightly lower . SimilarThis study has several limitations. The cross-sectional design limits the ability to assess causation, and longitudinal studies are needed to determine causality. To assess steatosis and significant fibrosis, we relied on non-invasive tests (NITs), although liver biopsy currently remains the reference standard to define hepatic fibrosis . On the This study identified several predictors of poor HRQL in PLWH. Moreover, PLWH and significant fibrosis may reflect a population at risk that need closer monitoring to improve liver-related and patient-reported outcomes. Targeted interventions and counseling in patients with hepatic steatosis and fibrosis could be an aspect of overcoming the impaired HRQL in PLWH.Supplementary file1 (DOCX 29 kb)Below is the link to the electronic supplementary material.


Outcomes of interest were (sepsis-related) mortality, sepsis severity ), sepsis testing, and antibiotic usage. Differences before and after implementation of the guideline were assessed using logistic and linear regression analysis for binary and continuous outcomes respectively. All analyses were adjusted for gestational age and sex. Mortality within 10 days of a sepsis episode occurred in 39 (10.3%) and 34 (7.6%) episodes in the pre- and post-implementation period respectively (P = 0.13). The nSOFA course during a sepsis episode was significantly lower in the post-implementation group (P = 0.01). We observed significantly more blood tests for determination of inflammatory biomarkers, but no statistically significant difference in number of blood cultures drawn and in antibiotic usage between the two periods.The aim of this study was to investigate the association between the implementation of a local heart rate variability (HRV) monitoring guideline combined with determination of inflammatory biomarkers and mortality, measures of sepsis severity, frequency of sepsis testing, and antibiotic usage, among very preterm neonates. In January 2018, a guideline was implemented for early detection of late-onset neonatal sepsis using HRV monitoring combined with determination of inflammatory biomarkers. Data on all patients admitted with a gestational age at birth of < 32 weeks were reviewed in the period January 2016–June 2020 (Conclusion: Implementing HRV monitoring with determination of inflammatory biomarkers might help identify patients with sepsis sooner, resulting in reduced sepsis severity, without an increased use of antibiotics or number of blood cultures. Neonatal sepsis is a major health issue, particularly in preterm neonates, where low gestational age, low birth weight, immature immune system, and other compromising factors make it an important cause of morbidity and death –5. Late-To address this concern, we implemented as a standard of care a HRV monitoring guideline in which we, based on changes in HRV, determine interleukin-6 (IL-6), procalcitonin (PCT), and C-reactive protein (CRP) to diagnose LONS see Fig. . In appaThe study was conducted at the Erasmus MC University Medical Center–Sophia Children’s Hospital Rotterdam, a level-IV NICU. Electronic health record data from January 2016 until December 2017 were reviewed for all neonates with a gestational age below 32 weeks, who got admitted to the NICU. Data from January 2018 until June 2020 were available in a prospectively collected database.HRV monitoring ; Charlottesville, Virginia)) was introduced in January 2018. The HeRO monitor displays a HRC index which is calculated every hour and ranges between 0 and 7. This number reflects the fold increase in risk of sepsis in the next 24 h. Until January 2018, clinical staff was blinded for the HRC index. We developed a local guideline for preterm neonates older than 72 h , and CRP  levels at moment of sepsis suspicion were queried from the laboratory information system.The department of Microbiology at the Erasmus MC provided all results of blood cultures, including timing of collection and identified micro-organisms. One patient could provide multiple cases of suspected LONS. LONS diagnosis was established by the criteria defined by the NICHD Neonatal Research Network . An epis9 cells/L.The outcomes of interest were as follows: overall-mortality, 10-day mortality after sepsis suspicion, and measures of sepsis severity; neonatal Sequential Organ Failure Assessment (nSOFA) score , need foU test for continuous data and the chi-square test for categorical data. A logistic regression analysis was used to investigate the difference in binary outcome measures, and a linear regression was used to investigate the difference in continuous outcome measures before and after implementation of the HRV guideline combined with biomarker screening. All analyses were adjusted for gestational age and sex. Additional sensitivity analysis adjusting for underlying trends in treatment strategies was performed. A generalized linear model was built to analyze differences in the nSOFA course during the sepsis course in patients with sepsis. For all tests, a P-value of < 0.05 was considered significant.Statistical analysis was performed using SPSS  and R (R Core Team (2017), Vienna, Austria). Categorical variables were described using absolute numbers and frequencies. Continuous variables were described using medians and interquartile range because of non-normal distributions. Pre-post implementation comparisons were performed using the Mann–Whitney In the pre-implementation period, a total of 515 patients (< 32 weeks GA) were admitted to the NICU, in the post-implementation period 620 patients. In the study period, a total of 811 blood cultures in 473 prematurely born neonates (< 32 weeks GA) were withdrawn. Of these episodes, 490 (60.4%) were classified as sepsis with a similar distribution of type of sepsis (see Table P = 0.70). In the pre-implementation period, death within 10 days after blood culture occurred in 39 (10.3%) episodes and in the post-implementation period in 34 (7.6%) episodes (adjusted odds ratio (aOR) 0.69 (95% CI 0.42–1.12) after implementation, P = 0.13) .Figure P = 0.75). Patients needed intubation in 73 episodes (26.0%) and in 66 (21.0%) episodes in the pre-implementation and in the post-implementation period, respectively (aOR 0.71 (95% CI 0.48–1.04) after implementation, P = 0.08). Patients had severe thrombocytopenia in 52 episodes (13.8%) and in 37 episodes (8.5%) in the pre-implementation and in the post-implementation period, respectively (21.0%) (aOR 0.57 (95% CI 0.36–0.90) after implementation, P = 0.02) (see Table Patients needed inotropic support in 65 episodes 17.2%) and in 74 episodes (17.1%) episodes in the pre-implementation and in the post-implementation period, respectively (aOR 0.94 (95% CI 0.65–1.36) after implementation, % and in P = 0.51). The median number of blood withdrawals for inflammatory biomarkers was 1 in the pre-implementation period and it was 3 in the post-implementation period (P < 0.001) (see Table The median number of blood cultures drawn per patient in the pre-implementation period was 2 and in the post-implementation period it was 2 (P = 0.91). Pre-implementation the number of antibiotic days over hospitalization was 178.6 per 1000 days and post-implementation this was 168.1 per 1000 days (P = 0.06)  in a randomized controlled trial including over 3,000 preterm neonates [Our results are in line with the study of Moorman et al. which showed that implementing HRV monitoring led to mortality reduction  treatment. PTX is a phosphodiesterase inhibitor, originally registered for intermittent claudication in adults , which sAdditionally, our PTX protocol is an example on how to use an early warning system followed by determination of inflammatory biomarkers that give information about the subsequent disease course, for practicing pro-active personalized treatment , 33.Our study has some limitations. We performed a cohort study including all patients with a gestational age < 32 weeks admitted to our hospital. This type of study does not allow for a concurrent control group, limiting our ability to attribute causation. We obtained many variables and outcome data from clinical charts, which may lead to misclassification due to mistakes in data entry. If misclassification would occur, we would expect this to be random decreasing precision of the effect estimates. We tried to limit missing data and misclassification by double data entry and using automated registries for blood cultures from the department of microbiology.Also, although the current study reports on one of the largest cohorts with HRV monitoring, the sample size is still relatively small. The number needed to monitor for saving one life, reported by Moorman et al., is 23 patients . The nonIn conclusion, our findings show a trend towards lower 10-day mortality after sepsis suspicion and a significant reduction of sepsis severity after implementation of HRV monitoring combined with determination of inflammatory biomarkers. We observed no increase in antibiotic usage and the frequency of sepsis testing. However, this guideline logically was associated with an increase in blood withdrawals to determine inflammatory biomarkers. Increasing our understanding in HRV monitoring could potentially help reduce this. Implementing HRV monitoring with determination of inflammatory biomarkers might help identify patients with sepsis sooner, resulting in reduced sepsis severity, without an increased risk of increased antibiotic usage and sepsis testing.