Supplementary Material for: CD4+CD25+ Regulatory T Cells Inhibit Natural Killer Cell Hepatocytotoxicity of Hepatitis B Virus Transgenic Mice via Membrane-Bound TGF-β and OX40

CD4+CD25+ regulatory T cells (Tregs) are involved in the regulation of physiological and pathological hepatic immune responses, but the roles are not well explored in natural killer (NK) cell-mediated liver diseases. In this study, using the NK cell-mediated oversensitive liver injury model of hepatitis B virus transgenic (HBs-Tg) mice triggered by a low dose of concanavalin A, it was observed that an increased number of CD4+CD25+Foxp3+ Tregs were accumulated in the liver, along with the recovery of liver injury. Adoptive transfer of hepatic Tregs from HBs-Tg mice but not wild B6 mice could significantly attenuate the oversensitive liver injury via inhibiting liver accumulation and decreasing NK cell group 2D-mediated activation of NK cells in the recipient HBs-Tg mice. Furthermore, upregulated expression of membrane-bound TGF-β (mTGF-β) and OX40 on hepatic Tregs were demonstrated to account for inhibiting the NK cell-mediated hepatic injury in HBs-Tg mice through cell-cell contact, confirmed by antibody blockade and cell Transwell experiments in vivo and in vitro<i>. </i>Our findings for the first time indicated that CD4+CD25+ Tregs directly suppressed NK cell-mediated hepatocytotoxicity through mTGF-β and OX40/OX40L interaction in a cell-cell contact manner in HBV-associated liver disease

Supplementary Material for: Incidence of Respiratory Viral Infections Detected by PCR and Real-Time PCR in Adult Patients with Community-Acquired Pneumonia: A Meta-Analysis

<b><i>Background:</i></b> With the development of more rapid and sensitive detection methods based on PCR techniques, the contributions of respiratory viral infections to community-acquired pneumonia (CAP) in adult patients are being more and more recognized. Yet, up to now, there has been a lack of synthetic data that clearly demonstrates the incidence of respiratory viral infections in adult patients with CAP. <b><i>Objectives:</i></b> We intended to demonstrate the incidence of respiratory viral infections detected by PCR and real-time PCR in adult patients with CAP. <b><i>Methods:</i></b> We searched PubMed and Embase for studies providing the incidence of respiratory viral infections in adult patients with CAP. We investigated potential sources of heterogeneity by a univariant metaregression analysis and calculated the combined incidence of viral infections, viral infections mixed with other pathogens and individual respiratory virus species. <b><i>Results:</i></b> We eventually identified 23 eligible reports with a total number of 6,404 patients. Incidences ranged from 8.6 to 56.2% for overall respiratory viral infections. We noted significant heterogeneity in incidence estimates for the incidence of viral infections (Cochran's χ² = 269.9, p < 0.0001, I² = 91.8%). The combined incidence of viral infections was 22.4% (95% CI = 19.0-25.7). Incidences of viral coinfections with other pathogens ranged from 3 to 28%. A high level of heterogeneity was identified as well during the estimates for incidences of coinfections (χ² = 200.9, p < 0.0001, I² = 91.5%). The combined incidence of viral coinfections with other pathogens was 12.4% (95% CI = 9.7-15.0). Our heterogeneity analyses suggested that a lower respiratory tract sample was associated with higher overall viral incidence. Moreover, the influenza virus, rhinovirus and coronavirus were the 3 most frequently detected viral pathogens in adult patients with CAP according to our study. <b><i>Conclusions:</i></b> Respiratory viruses are probably crucial pathogens of adult patients with CAP, with the influenza virus being the most frequent viral pathogen identified. More than half of the viral infections are characterized as mixed infections with other pathogens

Supplementary Material for: A Novel Phytase Derived from an Acidic Peat-Soil Microbiome Showing High Stability under Acidic Plus Pepsin Conditions

Four novel phytases of the histidine acid phosphatase family were identified in two publicly available metagenomic datasets of an acidic peat-soil microbiome in northeastern Bavaria, Germany. These enzymes have low similarity to all the reported phytases. They were overexpressed in <i>Escherichia coli</i> and purified. Catalytic efficacy in simulated gastric fluid was measured and compared among the four candidates. The phytase named rPhyPt4 was selected for its high activity. It is the first phytase identified from unculturable Acidobacteria. The phytase showed a longer half-life than all the gastric-stable phytases that have been reported to date, suggesting a strong resistance to low pH and pepsin. A wide pH profile was observed between pH 1.5 and 5.0. At the optimum pH (2.5) the activity was 2,790 μmol/min/mg at the physiological temperature of 37°C and 3,989 μmol/min/mg at the optimum temperature of 60°C. Due to the competent activity level as well as the high gastric stability, the phytase could be a potential candidate for practical use in livestock and poultry feedin

Supplementary Material for: Adolescence predatory risk alters social behaviors and cognitive ability and central oxytocin and vasopressin expression in adult Brandt’s voles

Introduction: Stress during adolescence causes long-term behavioral changes in adulthood. We previously found that adolescent exposure to predatory risk augments adolescent social contact and adult parental behavior in Brandt’s voles (Lasiopodomys brandtii). Methods: Here we determined whether this experience alters sexual behavior, pair-bond formation, and recognition ability, as well as basal HPA axis activity, central oxytocin (OT), and arginine-vasopressin (AVP) expression in adulthood. Results: In the social interaction test, repeated cat odor (CO) exposure enhanced the frequency of lordosis by female voles toward an unfamiliar opposite-sex conspecific. CO voles preferred to engage with their partners after 48 h cohabitation whereas the control groups did not, which may reflect stable pair bonds in the CO treatment group. Furthermore, adolescent exposure to CO inhibited novel object and place recognition ability, while it influenced social recognition only among adult males. No effect of adolescent CO exposure was observed for basal HPA axis activity, showing a habituation effect. Finally, we found that CO exposure increased OT and decreased AVP expression in the hypothalamus, including the paraventricular nucleus and anterior hypothalamus. The levels of OT in the medial amygdala were lower, and AVP in the lateral septum was higher in CO voles compared with the control. Discussion/Conclusion: These findings demonstrate that adolescent exposure to predator risk promotes adult reproductive behavior of Brandt’s voles. Deficits in recognition ability may necessitate alterations in reproductive strategies to enhance inclusive fitness. OT and AVP systems may play a modulatory role in the alteration of social behaviors elicited by adolescent predatory risk

Erratum: Clinicopathological Features and Therapeutic Responses of Chinese Patients with Advanced Lung Adenocarcinoma Harboring an Anaplastic Lymphoma Kinase Rearrangement

<b><i>Background:</i></b> Presence of anaplastic lymphoma kinase (<i>ALK</i>) rearrangement is an indication for crizotinib in the treatment of patients with advanced or metastatic lung adenocarcinoma. Here, we sought to elucidate the association between clinicopathological features and <i>ALK</i> rearrangement status in Chinese patients with advanced lung adenocarcinoma harboring an <i>ALK</i> rearrangement. <b><i>Patients and Methods:</i></b><i>ALK</i> rearrangement status was determined using immunohistochemistry (IHC) in tumor tissues from 120 patients with advanced lung adenocarcinoma, and further assessed by fluorescence in situ hybridization (FISH) assay. The associations between <i>ALK</i> rearrangement status and clinicopathological features were analyzed. <b><i>Results:</i></b> According to IHC testing, the <i>ALK</i>-positive rate among the advanced lung adenocarcinoma patients was 6.67% (8/120). FISH validation found 5 patients with <i>ALK</i> rearrangement among the 8 IHC-positive cases. No significant difference was observed regarding age, sex, or smoking status between FISH-positive and -negative patients (p > 0.05). None of the 5 FISH-positive patients benefited from first-line chemotherapy. <b><i>Conclusion:</i></b> IHC can be used as a reliable method for <i>ALK</i> rearrangement screening in patients with lung adenocarcinoma, but further FISH validation is imperative. Presence of <i>ALK</i> rearrangement predicts a more aggressive biological behavior of the tumor and might be indicative of poor response to chemotherapy

Supplementary Material for: Ruyi Zhenbao Tablet and Baimai Ointment Therapy in Acute Ischemic Stroke: A Study Protocol for a Multi-center, Randomized, Double-blinded, and Parallel Controlled Trial

Introduction: Stroke is characterized by high incidence, recurrence rate, and mortality. Patients with acute is-chemic stroke (AIS) who are ineligible for acute revascularisation therapy require more effective medication treatments. A previous clinical study showed that Ruyi Zhenbao tablets and Baimai ointments might be effective against AIS; however, high-quality clinical evidence supporting their application in AIS is lacking. To explore the efficacy of the two classic Tibetan medicines in the treatment of AIS, a randomized clinical trial will be conducted in patients with AIS who are not eligible for thrombolytic treatment. Methods: A prospective, randomized, multiple-center, double-blinded, placebo-controlled, and parallel-group trial will be conducted. We shall randomize 480 eligible participants to either the intervention or the control group. The distribution ratio of each group will be 1:1:1:1, including 120 patients each in the dual-medication group, the Baimai ointment group, the Ruyi Zhenbao tablet group, and the placebo group. Participants will be treated with medication for 8 weeks, and they will receive three follow-up visits: at 4 weeks(D29), 8 weeks(D56), and 90 days (D90) after commencing treatment. The primary outcome will be D90 change in the simplified Fugl-Meyer score from baseline to post-treatment. The secondary outcomes are as follows:D29 change of simplified Fugl -Meyer score from baseline to post-treatment; Proportion of participants whose D29 NIHSS scores decreased by four or more points from baselineD90 proportion of subjects with mRS score of 0-2 (inclu-sive); D90 proportion of subjects with Barthel index score ≥95; D90 incidence of cardiovascular and cerebro-vascular events. Safety endpoint includes:Mortality within 90 days;Proportion of subjects with adverse events/ serious adverse events within 90 days. Conclusion: This research protocol lays a solid groundwork for its practical execution. This study is poised to serve as a reference for other Tibetan medicine researchers, contributing to the reduction of stroke-related expendi-tures globally and, in turn, benefiting a broader population of stroke patients

Supplementary Material for: Comparison of Stromal/Stem Cells Isolated from Human Omental and Subcutaneous Adipose Depots: Differentiation and Immunophenotypic Characterization

The emerging field of regenerative medicine has identified adipose tissue as an abundant source of stromal/stem cells for tissue engineering applications. Therefore, we have compared the differentiation and immunophenotypic features of adipose-derived stromal/stem cells (ASC) isolated from either omental or subcutaneous adipose depots.<b> </b>Human tissue samples were obtained from bariatric and plastic surgical practices at a university-affiliated teaching hospital and a private practice, respectively, with informed patient consent.<b> </b>Primary cultures of human ASC were isolated from adipose specimens within 24 h of surgery and culture expanded in vitro. The passaged ASC were induced to undergo adipogenic or osteogenic differentiation as assessed by histochemical methods or evaluated for surface antigen expression profiles by flow cytometry. ASC yields per unit weight of tissue were comparable between omental and subcutaneous depots. At passage 0, the immunophenotype of omental and subcutaneous ASC were not significantly different with the exception of CD105 and endoglin, a component of the transforming growth factor β receptor. The adipogenic differentiation of omental ASC was less robust than that of subcutaneous ASC based on in vitro histochemical and PCR assays. Although the yield and immunophenotype of ASC from omental adipose depots resembled that of subcutaneous ASC, omental ASC displayed significantly reduced adipogenic differentiation capacity following chemical induction. Further studies are necessary to evaluate and optimize the differentiation function of omental ASC in vitro and in vivo. Pending such analyses, omental ASC should not be used interchangeably with subcutaneous ASC for regenerative medical applications

Supplementary Material for: Preoperative H. pylori eradication therapy facilitates precise delineation in early gastric cancer with current H. pylori infection

[Introduction] Early gastric cancer with current Helicobacter pylori infection (HpC-EGC) is common but still unclear whether H. pylori eradication therapy (Hp-ET) or endoscopic submucosal dissection (ESD) should be performed first. We evaluated Hp-ET’s short-term effects on horizontal boundary delineations of HpC-EGC in ESD. [Methods] Prospectively enrolled HpC-EGC patients were randomly assigned to eradication or control groups. Operation scopes of HpC-EGC lesion were delineated with marking dots at 5 mm out of the endoscopic demarcation line by an independent endoscopist, unaware of eradication status, before formal circumferential incision. As representatives, precise delineation rate, the shortest distance of all marking dots to the pathological demarcation line in all slices of one intact resected specimen (Dmin), and negative marking dot specimen rate were examined. [Results] 23 HpC-EGC patients (25 lesions) were allocated to eradication group and 26 patients (27 lesions) were allocated to control group with similar eradication success rates and all were differentiated type. With improving background mucosa inflammation after Hp-ET and similar gastritis-like epithelium rates, 10 lesions (40.0%) in eradication group were of precise delineation compared to control group with 2 lesions (7.4%) (RR = 5.40, 95% CI 1.31-22.28). Dmin of eradication and control groups were 4.17 ± 2.52 mm and 2.67 ± 2.30 mm (p = 0.029), accompanied by 4 (14.8%) and none (0.0%) specimens that exhibited positive marking dots (p = 0.11), respectively. [Conclusion] For HpC-EGC patients, administrating eradication medication before ESD is beneficial for the precise delineation of lesions and reducing the risk of positive horizontal resection margins

Supplementary Material for: Preoperative H. pylori eradication therapy facilitates precise delineation in early gastric cancer with current H. pylori infection

[Introduction] Early gastric cancer with current Helicobacter pylori infection (HpC-EGC) is common but still unclear whether H. pylori eradication therapy (Hp-ET) or endoscopic submucosal dissection (ESD) should be performed first. We evaluated Hp-ET’s short-term effects on horizontal boundary delineations of HpC-EGC in ESD. [Methods] Prospectively enrolled HpC-EGC patients were randomly assigned to eradication or control groups. Operation scopes of HpC-EGC lesion were delineated with marking dots at 5 mm out of the endoscopic demarcation line by an independent endoscopist, unaware of eradication status, before formal circumferential incision. As representatives, precise delineation rate, the shortest distance of all marking dots to the pathological demarcation line in all slices of one intact resected specimen (Dmin), and negative marking dot specimen rate were examined. [Results] 23 HpC-EGC patients (25 lesions) were allocated to eradication group and 26 patients (27 lesions) were allocated to control group with similar eradication success rates and all were differentiated type. With improving background mucosa inflammation after Hp-ET and similar gastritis-like epithelium rates, 10 lesions (40.0%) in eradication group were of precise delineation compared to control group with 2 lesions (7.4%) (RR = 5.40, 95% CI 1.31-22.28). Dmin of eradication and control groups were 4.17 ± 2.52 mm and 2.67 ± 2.30 mm (p = 0.029), accompanied by 4 (14.8%) and none (0.0%) specimens that exhibited positive marking dots (p = 0.11), respectively. [Conclusion] For HpC-EGC patients, administrating eradication medication before ESD is beneficial for the precise delineation of lesions and reducing the risk of positive horizontal resection margins