5-Hydroxytryptamine Enhances the Pacemaker Activity of Interstitial Cells of Cajal in Mouse Colon.

We examined the localization of the 5-hydroxytryptamine (5-HT) receptor and its effects on mouse colonic interstitial cells of Cajal (ICCs) using electrophysiological techniques. Treatment with 5-HT increased the pacemaker activity in colonic ICCs with depolarization of membrane potentials in a dose-dependent manner. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers blocked pacemaker activity and 5-HT-induced effects. Moreover, an adenylate cyclase inhibitor inhibited 5-HT-induced effects, and cell-permeable 8-bromo-cAMP increased the pacemaker activity. Various agonists of the 5-HT receptor subtype were working in colonic ICCs, including the 5-HT4 receptor. In small intestinal ICCs, 5-HT depolarized the membrane potentials transiently. Adenylate cyclase inhibitors or HCN blockers did not show any influence on 5-HT-induced effects. Anoctamin-1 (ANO1) or T-type Ca2+ channel blockers inhibited the pacemaker activity of colonic ICCs and blocked 5-HT-induced effects. A tyrosine protein kinase inhibitor inhibited pacemaker activity in colonic ICCs under controlled conditions but did not show any influence on 5-HT-induced effects. Among mitogen-activated protein kinase (MAPK) inhibitors, a p38 MAPK inhibitor inhibited 5-HT-induced effects on colonic ICCs. Thus, 5-HT's effect on pacemaker activity in small intestinal and colonic ICCs has excitatory but variable patterns. ANO1, T-type Ca2+, and HCN channels are involved in 5-HT-induced effects, and MAPKs are involved in 5-HT effects in colonic ICCs.

Parallel accumulation-serial fragmentation method for in-depth proteomic analysis of bronchoalveolar lavage fluid collected from patients with nonsmall cell lung cancer.

PURPOSE: Advances in mass spectrometry-based quantitative proteomic analysis have successfully demonstrated the in-depth detection of protein biomarkers in bronchoalveolar lavage fluid (BALF) from patients with lung cancers. Recently, ion mobility technology was incorporated into the mass spectrometers escalating the sensitivity and throughput. Utilizing these advantages, herein, we employed the parallel accumulation-serial fragmentation (PASEF) implanted in a timsTOF Pro mass spectrometer to examine the alteration of BALF proteomes in patients with nonsmall cell lung cancers (NSCLCs). EXPERIMENTAL DESIGN: BALF proteins were processed from patients with NSCLC and analyzed in a timsTOF Pro mass spectrometer with the PASEF method using a peptide input of 100 ng. Label-free mass spectrometry data were analyzed in the FragPipe platform. RESULTS: We quantitated over 1400 proteins from a single injection of 100 ng of peptides per sample with a median of ∼2000 proteins. We were able to find a few potential biomarker proteins upregulated in NSCLC. CONCLUSIONS AND CLINICAL RELEVANCE: The alterations of the BALF proteome landscape vary among patients with NSCLC as previously observed in patients with small-cell lung cancers. The PASEF method has significantly enhanced the sensitivity and throughput, demonstrating its effectiveness in clinical research and application.

The Immune Suppressor IGSF1 as a Potential Target for Cancer Immunotherapy.

The development of first-generation immune-checkpoint inhibitors targeting PD-1/PD-L1 and CTLA-4 ushered in a new era in anticancer therapy. Although immune-checkpoint blockade therapies have shown clinical success, a substantial number of patients yet fail to benefit. Many studies are under way to discover next-generation immunotherapeutic targets. Immunoglobulin superfamily member 1 (IGSF1) is a membrane glycoprotein proposed to regulate thyroid function. Despite containing 12 immunoglobin domains, a possible role for IGSF1, in immune response, remains unknown. Here, our studies revealed that IGSF1 is predominantly expressed in tumors but not normal tissues, and increased expression is observed in PD-L1low non-small cell lung cancer (NSCLC) cells as compared with PD-L1high cells. Subsequently, we developed and characterized an IGSF1-specific human monoclonal antibody, WM-A1, that effectively promoted antitumor immunity and overcame the limitations of first-generation immune-checkpoint inhibitors, likely via a distinct mechanism of action. We further demonstrated high WM-A1 efficacy in humanized peripheral blood mononuclear cells (PBMC), and syngeneic mouse models, finding additive efficacy in combination with an anti-PD-1 (a well-characterized checkpoint inhibitor). These findings support IGSF1 as an immune target that might complement existing cancer immunotherapeutics.

Comparative analysis of the pig gut microbiome associated with the pig growth performance.

There are a variety of microorganisms in the animal intestine, and it has been known that they play important roles in the host such as suppression of potentially pathogenic microorganisms, modulation of the gut immunity. In addition, the gut microbiota and the livestock growth performance have long been known to be related. Therefore, we evaluated the interrelation between the growth performance and the gut microbiome of the pigs from 3 different farms, with pigs of varied ages ready to be supplied to the market. When pigs reached average market weight of 118 kg, the average age of pigs in three different farms were < 180 days, about 190 days, and > 200 days, respectively. Fecal samples were collected from pigs of age of 70 days, 100 days, 130 days, and 160 days. The output data of the 16S rRNA gene sequencing by the Illumina Miseq platform was filtered and analyzed using Quantitative Insights into Microbial Ecology (QIIME)2, and the statistical analysis was performed using Statistical Analysis of Metagenomic Profiles (STAMP). The results of this study showed that the gut microbial communities shifted as pigs aged along with significant difference in the relative abundance of different phyla and genera in different age groups of pigs from each farm. Even though, there was no statistical differences among groups in terms of Chao1, the number of observed operational taxonomic units (OTUs), and the Shannon index, our results showed higher abundances of Bifidobacterium, Clostridium and Lactobacillus in the feces of pigs with rapid growth rate. These results will help us to elucidate important gut microbiota that can affect the growth performance of pigs.

Targeting isoforms of RON kinase (MST1R) drives antitumor efficacy.

Recepteur d'origine nantais (RON, MST1R) is a single-span transmembrane receptor tyrosine kinase (RTK) aberrantly expressed in numerous cancers, including various solid tumors. How naturally occurring splicing isoforms of RON, especially those which are constitutively activated, affect tumorigenesis and therapeutic response, is largely unknown. Here, we identified that presence of activated RON could be a possible factor for the development of resistance against anti-EGFR (cetuximab) therapy in colorectal cancer patient tissues. Also, we elucidated the roles of three splicing variants of RON, RON Δ155, Δ160, and Δ165 as tumor drivers in cancer cell lines. Subsequently, we designed an inhibitor of RON, WM-S1-030, to suppress phosphorylation thereby inhibiting the activation of the three RON variants as well as the wild type. Specifically, WM-S1-030 treatment led to potent regression of tumor growth in solid tumors expressing the RON variants Δ155, Δ160, and Δ165. Two mechanisms for the RON oncogenic activity depending on KRAS genotype was evaluated in our study which include activation of EGFR and Src, in a trimeric complex, and stabilization of the beta-catenin. In terms of the immunotherapy, WM-S1-030 elicited notable antitumor immunity in anti-PD-1 resistant cell derived mouse model, likely via repression of M1/M2 polarization of macrophages. These findings suggest that WM-S1-030 could be developed as a new treatment option for cancer patients expressing these three RON variants.

Implication of dental insurance status on patterns of pre-radiation dental extraction and risk of osteoradionecrosis of the jaw in head-and-neck cancer patients.

Oral toxicities such as osteoradionecrosis can be minimized by dental screening and prophylactic dental care prior to head and neck (HN) radiation therapy (RT). However, limited information is available about how dental insurance interacts with prophylactic dental care and osteoradionecrosis. To address this gap in knowledge, we conducted a cohort study of 2743 consecutive adult patients treated with curative radiation for HN malignancy who underwent pre-radiation dental assessment and where required, prophylactic dental treatment. Charts were reviewed to determine patient demographics, dental findings, dental treatment and development of osteoradionecrosis following radiation. Three insurance cohorts were identified: private-insured (50.4 %), public-insured (7.3 %), being patients with coverage through government-funded disability and welfare programs, and self-pay (42.4 %). More than half the public-insured patients underwent prophylactic pre-radiation dental extractions, followed by self-pay patients (44 %) and private-insured patients (26.6 %). After a median follow-up time of 4.23 years, 6.5 % of patients developed osteoradionecrosis. The actuarial rate of osteoradionecrosis in the public-insured patients was 14.7 % at 5-years post-RT, compared to 7.5 % in private-insured patients and 6.7 % in self-pay patients. On multivariable analysis, dental insurance status, DMFS160, age at diagnosis, sex, tumor site, nodal involvement, years smoked and gross income were all significant risk factors for tooth removal prior to HN radiation. However, only public-insured status, tumor site and years smoked were significant risk factors for development of osteoradionecrosis. Our findings demonstrate that lack of comprehensive dental coverage (patients who self-pay or who have limited coverage under public-insured programs) associates strongly with having teeth removed prior to HN RT. Nearly 1 in 6 patients covered under public-insurance developed osteoradionecrosis within 5 years of completing their treatment. Well-funded dental insurance programs for HN cancer patients might reduce the number of pre-RT extractions performed in these patients, improving quality of life post-RT.

A rapid assay for assessing bacterial effects on Arabidopsis thermotolerance.

BACKGROUND: The role of beneficial microbes in mitigating plant abiotic stress has received considerable attention. However, the lack of a reproducible and relatively high-throughput screen for microbial contributions to plant thermotolerance has greatly limited progress in this area, this slows the discovery of novel beneficial isolates and the processes by which they operate. RESULTS: We designed a rapid phenotyping method to assess the effects of bacteria on plant host thermotolerance. After testing multiple growth conditions, a hydroponic system was selected and used to optimize an Arabidopsis heat shock regime and phenotypic evaluation. Arabidopsis seedlings germinated on a PTFE mesh disc were floated onto a 6-well plate containing liquid MS media, then subjected to heat shock at 45 °C for various duration. To characterize phenotype, plants were harvested after four days of recovery to measure chlorophyll content. The method was extended to include bacterial isolates and to quantify bacterial contributions to host plant thermotolerance. As an exemplar, the method was used to screen 25 strains of the plant growth promoting Variovorax spp. for enhanced plant thermotolerance. A follow-up study demonstrated the reproducibility of this assay and led to the discovery of a novel beneficial interaction. CONCLUSIONS: This method enables rapid screening of individual bacterial strains for beneficial effects on host plant thermotolerance. The throughput and reproducibility of the system is ideal for testing many genetic variants of Arabidopsis and bacterial strains.

The prognostic impact of reduced variant burden in elderly patients with acute myeloid leukemia treated with decitabine.

BACKGROUND/AIMS: We evaluated the role of next-generation sequencing (NGS)-based disease monitoring for elderly patients diagnosed with acute myeloid leukemia (AML) who received decitabine therapy. METHODS: A total of 123 patients aged > 65 years with AML who received decitabine were eligible. We analyzed the dynamics of variant allele frequency (VAF) in 49 available follow-up samples after the fourth cycle of decitabine. The 58.6% VAF clearance (Δ, [VAF at diagnosis - VAF at follow-up] × 100 / VAF at diagnosis) was the optimal cut-off for predicting overall survival (OS). RESULTS: The overall response rate was 34.1% (eight patients with complete remission [CR], six of CR with incomplete hematologic recovery, 22 with partial responses, and six with morphologic leukemia-free status). Responders (n = 42) had significantly better OS compared with non-responders (n = 42) (median, 15.3 months vs. 6.5 months; p < 0.001). Of the 49 patients available for follow-up targeted NGS analysis, 44 had trackable gene mutations. The median OS of patients with ΔVAF ≥ 58.6% (n=24) was significantly better than that of patients with ΔVAF < 58.6% (n = 19) (20.5 months vs. 9.8 months, p = 0.010). Moreover, responders with ΔVAF ≥ 58.6% (n = 20) had a significantly longer median OS compared with responders with VAF < 58.6% (n = 11) (22.5 months vs. 9.8 months, p = 0.004). CONCLUSION: This study suggested that combining ΔVAF ≥ 58.6%, a molecular response, with morphologic and hematologic responses can more accurately predict OS in elderly AML patients after decitabine therapy.

Effect of adenosine-1 receptor activation on pacemaker activity of interstitial cells of Cajal from mouse colon.

Adenosine plays an important role on gastrointestinal (GI) motility through adenosine receptors. Interstitial cells of Cajal (ICC) are pacemaker cells that regulate GI smooth muscle activity. The functional role and its signal mechanism of adenosine on the pacemaker activity were investigated using whole-cell patch clamp, RT-PCR, and intracellular Ca2+-imaging with ICC from mouse colon. Adenosine depolarized the membrane potentials and increased the pacemaker potential frequency, which was blocked by a selective A1-receptor antagonist, but not A2a-, A2b, or A3-receptor antagonist. A selective A1 receptor agonist represented similar effects as those of adenosine and mRNA transcript of A1-receptor was expressed in ICC. The adenosine-induced effects were blocked by phospholipase C (PLC) and a Ca2+-ATPase inhibitor. Adenosine increased spontaneous intracellular Ca2+ oscillations, as seen fluo4/AM. Both hyperpolarization-activated cyclic nucleotide (HCN) channel inhibitors and adenylate cyclase inhibitors blocked the adenosine-induced effects. And adenosine increased the basal cellular adenylate cyclase activity in colonic ICC. However, adenosine and adenylate cyclase inhibitors did not show any influence on pacemaker activity in small intestinal ICC for a comparison with that of the small intestine. These results suggest adenosine modulates the pacemaker potentials by acting HCN channels- and intracellular Ca2+- dependent mechanisms through A1-receptor. Therefore, adenosine may be a therapeutic target in colonic motility disorders.

Quantitative proteomic analysis of bronchoalveolar lavage fluids from patients with small cell lung cancers.

PURPOSE: Small cell lung cancer (SCLC) is one of the malignant cancers with aggressive progression and poor prognosis. Bronchoalveolar lavage fluid (BALF) has been arising recently as a potential source of biomarkers for lung cancers. In this study, we performed quantitative BALF proteomic analysis to identify potential biomarkers for SCLC. EXPERIMENTAL DESIGN: BALF were collected from tumor-bearing lungs and non-tumor lungs of five SCLC patients. Then, BALF proteomes were prepared for a TMT-based quantitative mass spectrometry analysis. Differentially expressed proteins (DEP) were identified when considering individual variation. Potential SCLC biomarker candidates were validated by immunohistochemistry (IHC). A public database of multiple SCLC cell lines was used to evaluate the correlation of these markers with SCLC subtypes and chemo-drug responses. RESULTS: We identified 460 BALF proteins in SCLC patients and observed considerable individual variation among the patients. Immunohistochemical analysis and bioinformatics resulted in the identification of CNDP2 and RNPEP as potential subtype markers for ASCL1 and NEUROD1, respectively. In addition, CNDP2 was found to be positively correlated with responses to etoposide, carboplatin, and irinotecan. CONCLUSIONS AND CLINICAL RELEVANCE: BALF is an emerging source of biomarkers, making it useful for the diagnosis and prognosis of lung cancers. We characterized the proteomes of paired BALF samples collected from tumor-bearing and non-tumor lungs of SCLC patients. Several proteins were found elevated in tumor-bearing BALF, and especially CNDP2 and RNPEP appeared to be potential indicators for ASLC1-high and NEUROD1-high subtypes of SCLC, respectively. The positive correlation of CNDP2 with chemo-drug responses would help to make decisions for treatment of SCLC patients. These putative biomarkers could be comprehensively investigated for a clinical use towards precision medicine.

Prospective Cohort Study with a 2-Year Follow-up of Clinical Results, Fusion Rate, and Muscle Bulk for Uniportal Full Endoscopic Posterolateral Transforaminal Lumbar Interbody Fusion.

STUDY DESIGN: Retrospective cohort study. PURPOSE: Postoperative evaluation of the cross-sectional area of paraspinal muscle and clinical findings in patients who had interlaminar route uniportal full endoscopic posterolateral transforaminal lumbar interbody fusion (EPTLIF) after 2 years. OVERVIEW OF LITERATURE: There are limited short-term follow-up studies on efficacy, safety, and physiological changes with a 2-year follow-up. There is no study on paraspinal muscle cross-sectional area change in patients who had undergone uniportal EPTLIF. METHODS: We evaluated patients who underwent EPTLIF with a minimum 24-month follow-up. Clinical parameters of the Visual Analog Scale (VAS) and Oswestry Disability Index (ODI) were measured at the preoperative, 1-week postoperative mark, postoperative 3-month mark, and final follow-up. Preoperative and 1-year postoperative magnetic resonance imaging measurement of preoperative and postoperative Kjaer grade, right and left psoas muscle mass area, and right and left paraspinal muscle mass area was performed. RESULTS: EPTLIF with a minimum 24-month follow-up of 35 levels was included. The complication rate was 6%, and the mean Bridwell's fusion grade was 1.37 (1-2). There was statistically significant improvement at 1 week, 3 months, and 2 years in VAS (4.11±1.23, 4.94±1.30, and 5.46±1.29) and in ODI (40.34±10.06, 46.69±9.14, and 49.63±8.68), respectively (p <0.05). Successful operation rate with excellent and good MacNab's criteria at 2 years was 97%. There was an increment of statistically significant bilateral psoas muscle cross-sectional area, right side (70.03±149.1 mm²) and left side (67.59±113.2 mm²) (p <0.05). CONCLUSIONS: Uniportal EPTLIF achieved good fusion and improved clinical outcomes with favorable paraspinal musculature bulk at the 2-year follow-up.

Remodeling of Epidural Fluid Hematoma after Uniportal Lumbar Endoscopic Unilateral Laminotomy with Bilateral Decompression: Comparative Clinical and Radiological Outcomes with a Minimum Follow-up of 2 Years.

STUDY DESIGN: Retrospective cohort study. PURPOSE: To evaluate the clinical and radiological effects of epidural fluid hematoma in the medium term after lumbar endoscopic decompression. OVERVIEW OF LITERATURE: There is limited literature comparing the effect of postoperative epidural fluid hematoma after uniportal endoscopic decompression. METHODS: Magnetic resonance imaging (MRI) and clinical evaluation were performed for patients with single-level uniportal endoscopic lumbar decompression with a minimum follow-up of 2 years. RESULTS: A total of 126 patients were recruited with a minimum follow-up of 26 months. The incidence of epidural fluid hematoma was 27%. Postoperative MRI revealed a significant improvement in the postoperative dura sac area at postoperative day 1 and at the upper endplate at 6 months in the hematoma cohort (39.69±15.72 and 26.89±16.58 mm2) as compared with the nonhematoma cohort (48.92±21.36 and 35.1±20.44 mm2), respectively (p <0.05); and at the lower endplate on postoperative 1 day in the hematoma cohort (51.18±24.69 mm2) compared to the nonhematoma cohort (63.91±27.92 mm2) (p <0.05). No significant difference was observed in the dura sac area at postoperative 1 year in both cohorts. The hematoma cohort had statistically significant higher postoperative 1-week Visual Analog Scale (VAS; 3.32±0.68) pain and Oswestry Disability Index (ODI; 32.65±5.56) scores than the nonhematoma cohort (2.99±0.50 and 30.02±4.84, respectively; p <0.05). No significant difference was found at the final follow-up VAS, ODI, and MRI dura sac area. CONCLUSIONS: Epidural fluid hematoma is a common early postoperative MRI finding in lumbar endoscopic unilateral laminotomy with bilateral decompression. Conservative management is the preferred treatment option for patients who do not have a neurological deficit. Symptoms last only a few days and are self-limiting. A common endpoint is a remodeled fluid hematoma and the subsequent expansion of the dura sac area.

Minimally Neural Retractive, Total Annular Resection, Transforaminal Endoscopic (TARTE) Approach for Severely Canal Compromising Lumbar Disk Herniation Accompanied by a Neurologic Deficit.

OBJECTIVE: Treatment of severely canal compromising lumbar disk herniations (LDH), occupying more than 50% of the canal area, are associated with technical difficulty and worse outcomes. This study aimed to describe new techniques of transforaminal endoscopic lumbar diskectomy (TELD) with less neural retraction, and total annular resection for broad-based severely canal compromising disk herniation. We also evaluated the feasibility and safety of the techniques, and analyzed the clinical and radiologic outcomes of 32 patients presenting with neurologic deficits. METHODS: A retrospective cohort study was performed with 32 consecutive patients who underwent TELD for broad-based severely canal compromising LDH between January 2018 and January 2020. We removed the LDH using two novel techniques: (1) the "mobile outside-in" approach and total annular resection method and (2) internal decompression and subsequent pushdown method of the migrated fragment. The cross-sectional area (CSA) on magnetic resonance image was evaluated preoperatively and compared with the postoperative image within 7 days and between 6 months and 1 year. The visual analog scale (VAS) for back and leg pain, Oswestry disability index (ODI), MacNab's criteria, and motor power of the involved lower extremities were evaluated pre- and postoperatively. RESULTS: A total of 32 patients, with an average age of 37.5 years (range: 17-66), underwent surgery. The mean VAS score for back pain improved from 7.84 ± 1.02 to 1.31 ± 0.54 and the ODI score improved from 74.3 ± 7.82 to 20.4 ± 3.71 at final follow-up. According to MacNab's criteria, 23 patients had excellent and 9 patients had good outcomes at final follow-up. All patients operated on at the L4-L5 level had great toe/ankle dorsiflexion and/or ankle plantar flexion weakness; knee extension weakness was found at the L2-L3 and L3-L4 levels. Motor function improved significantly; the mean values and range preoperatively, and at 1 month, 3 months, and final follow-up, were 3.41 ± 0.95 (1-4), 4.56 ± 0.56 (3-5), 4.88 ± 0.34 (4-5), and 4.97 ± 0.18 (4-5), respectively (p < 0.001, at all follow-up). The mean values and range of the preserved CSA proportion, preoperatively and within 1 week after surgery, and at final follow-up were 34.9 ± 10.9 (15-61), 81.06 ± 10.24 (63-97), and 93.03 ± 5.37 (76-99), respectively (p < 0.001, at all follow-up). CONCLUSION: The transforaminal endoscopic approach for broad-based severely canal compromising LDH can be considered a feasible surgical option for an experienced surgeon. With total annular resection and pushdown of migrated fragments, safe and complete removal of LDH was possible in patients with a neurologic deficit. Total annular resection may increase the overall but not the early recurrence rate.

Retrospective Case Control Study: Clinical and Computer Tomographic Fusion and Subsidence Evaluation for Single Level Uniportal Endoscopic Posterolateral Approach Transforaminal Lumbar Interbody Fusion Versus Microscopic Minimally Invasive Transforaminal Interbody Fusion.

STUDY DESIGN: Retrospective comparative study. OBJECTIVE: Assessment of difference in clinical and computer tomographic outcomes between the 2 cohorts. METHODS: Computer tomographic evaluation by Bridwell's grade, Kim's stage, Kim's subsidence grade and clinical evaluation by VAS, ODI and McNab's criteria on both cohorts. RESULTS: 33 levels of Endo-TLIF and 22 levels of TLIF were included, with a mean follow up of 14.3 (10-24) and 22.9 (13-30) months respectively. Both Endo-TLIF and TLIF achieved significant improvement of pain and ODI at post-operative 4 week, 3 months and at final follow up with VAS 4.39 ± 0.92, 5.27 ± 1.16 and 5.73 ± 1.21in Endo-TLIF and 4.55 ± 1.16, 5.05 ± 1.11 and 5.50 ± 1.20 in TLIF respectively and ODI at post-operative 1 week, 3 months and final follow up were 43.15 ± 6.57, 49.27 ± 8.24 and 51.73 ± 9.09 in Endo-TLIF and 41.73 ± 7.98, 46.18± 8.46 and 49.09 ± 8.98 in TLIF respectively, P < 0.05. Compared to TLIF, Endo-TLIF achieved better VAS with 0.727 ± 0.235 at 3 months and 0.727 ± 0.252 at final follow up and better ODI with 3.88 ± 1.50 at 3months and 3.42 ± 1.63 at final follow up, P < 0.05. At 6 months radiological evaluation comparison of the Endo-TLIF and TLIF showed significant with more favorable fusion rate in Endo-TLIF of -0.61 ± 0.12 at 6 months and -0.49 ± 0.12 at 1 year in Bridwell's grading and 0.70 ± 0.15 at 6 months and 0.56 ± 0.14 at 1 year in Kim's stage.There is less subsidence of 0.606 ± 0.18 at 6 months and -0.561 ± 0.20 at 1 year of Kim's subsidence grade, P < 0.05. CONCLUSION: Application of single level uniportal endoscopic posterolateral lumbar interbody fusion achieved better clinical outcomes and fusion rate with less subsidence than microscopic minimally invasive transforaminal lumbar interbody fusion in mid-term evaluation for our cohorts of patients.

Clinical Usefulness of Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Low Serum Testosterone Measurement.

Background: Mass spectrometry methods exhibit higher accuracy and lower variability than immunoassays at low testosterone concentrations. We developed and validated an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for quantifying serum total testosterone. Methods: We used an ExionLC UPLC (Sciex, Framingham, MA, USA) system and a Sciex Triple Quad 6500+ (Sciex) MS/MS system in electrospray ionization and positive ion modes with multiple reaction monitoring transitions to evaluate precision, accuracy, linearity, lower limit of quantitation (LLOQ), carryover, ion suppression, stability, and reference intervals. For method comparison, we measured serum testosterone concentrations using this method in 40 subjects whose testosterone concentrations ranged from 0.14 to 55.48 nmol/L as determined using the Architect i2000 immunoassay (Abbott Diagnostics, Abbott Park, IL, USA) and in an additional 160 sera with testosterone concentrations <1.67 nmol/L. Results: The intra- and inter-run precision CVs were <2.81%, and the accuracy bias values were <3.85%, which were all acceptable. The verified linear interval was 0.03-180.84 nmol/L; the LLOQ was 0.03 nmol/L. No significant carryover and ion suppression were observed. The testosterone in serum was stable at 4°C, at -20°C, and after three freeze-thaw cycles. The reference intervals were successfully verified. The correlation was good at testosterone concentrations of 0.14-55.48 nmol/L; however, the Architect assay showed positive percent bias at concentrations <1.67 nmol/L. Conclusions: The UPLC-MS/MS assay shows acceptable performance, with a lower LLOQ than the immunoassay. This method will enable the quantitation of low testosterone concentrations.

Performance of abiotic stress-inducible synthetic promoters in genetically engineered hybrid poplar (Populus tremula × Populus alba).

Abiotic stresses can cause significant damage to plants. For sustainable bioenergy crop production, it is critical to generate resistant crops to such stress. Engineering promoters to control the precise expression of stress resistance genes is a very effective way to address the problem. Here we developed stably transformed Populus tremula × Populus alba hybrid poplar (INRA 717-1B4) containing one-of-six synthetic drought stress-inducible promoters (SDs; SD9-1, SD9-2, SD9-3, SD13-1, SD18-1, and SD18-3) identified previously by transient transformation assays. We screened green fluorescent protein (GFP) induction in poplar under osmotic stress conditions. Of six transgenic lines containing synthetic promoter, three lines (SD18-1, 9-2, and 9-3) had significant GFP expression in both salt and osmotic stress treatments. Each synthetic promoter employed heptamerized repeats of specific and short cis-regulatory elements (7 repeats of 7-8 bases). To verify whether the repeats of longer sequences can improve osmotic stress responsiveness, a transgenic poplar containing the synthetic promoter of the heptamerized entire SD9 motif (20 bases, containing all partial SD9 motifs) was generated and measured for GFP induction under osmotic stress. The heptamerized entire SD9 motif did not result in higher GFP expression than the shorter promoters consisting of heptamerized SD9-1, 9-2, and 9-3 (partial SD9) motifs. This result indicates that shorter synthetic promoters (~50 bp) can be used for versatile control of gene expression in transgenic poplar. These synthetic promoters will be useful tools to engineer stress-resilient bioenergy tree crops in the future.

Effect of MED-02 Containing Two Probiotic Strains, Limosilactobacillus fermentum MG4231 and MG4244, on Body Fat Reduction in Overweight or Obese Subjects: A Randomized, Multicenter, Double-Blind, Placebo-Controlled Study.

MED-02 is a complex supplement containing two probiotic strains, Limosilactobacillus fermentum MG4231 and MG4244, isolated from humans. The anti-obesity effects and safety profile of MED-02 were assessed in overweight and obese subjects. In this randomized, double-blinded, placebo-controlled, multicenter study, 100 healthy obese and overweight subjects aged 19-65 years with a body mass index (BMI) between 25 and 31.9 kg/m2 were recruited and randomized to receive a placebo or MED-02 (5 × 109 CFU/day). After 12 weeks of consumption, body fat mass (-1166.82 g vs. -382.08 g; p = 0.024) and body fat percentage (-0.85% vs. -0.11%; p = 0.030), as evaluated by dual-energy X-ray absorptiometry (DEXA) and body weight (-2.06 kg vs. -1.22 kg; p = 0.041), were significantly reduced in the MED-02 group compared to the placebo group. The safety profile did not differ among the groups. No serious adverse effects were observed in either group. These results suggest that MED-02 is a safe and beneficial probiotics that reduces body fat and body weight in overweight or obese individuals.

Early gastric neoplasms are significant risk factor for colorectal adenoma: A prospective case-control study.

Although gastric cancer patients have a high incidence and risk of colorectal cancer, evidence is lacking regarding whether early gastric neoplasms (EGNs), such as gastric adenomas and early gastric cancer, are risk factors for colorectal adenoma. This study aimed to investigate the incidence of colorectal adenomas in patients with EGN. This prospective study was conducted between January 2015 and December 2016. Of the 307 patients who underwent gastric endoscopic submucosal dissection for EGN, 110 patients were enrolled in the EGN group, and 110 age- and sex-matched healthy persons from the screening population were included in the control group in a 1:1 ratio. Demographic factors and results of colonoscopy, including quality assessment, were collected, and analyzed. No significant differences in the quality of colonoscopy, including bowel preparation, cecal intubation rate, and withdrawal time between the 2 groups, were observed. The incidence of colorectal adenoma was significantly higher in the EGN group than in the control group (55.5% vs 26.4%, P = .001). Multivariate analysis confirmed that old age (odds ratio: 1.04, 95% confidence interval: 1.01-1.08, P = .005) and a history of EGN (odds ratio: 4.99, 95% confidence interval: 2.60-9.57, P = .001) were independent risk factors for colorectal adenoma. This is the first prospective study to reflect the quality indicator of colonoscopy and confirmed that old age and a history of EGN are significant risk factors for colorectal adenomas. Therefore, more stringent colonoscopy surveillance should be considered in elderly patients with EGN.

Investigation of the Hydrogen Sulfide Signaling Pathway in Schwann Cells during Peripheral Nerve Degeneration: Multi-Omics Approaches.

N-ethylmaleimide (NEM) inhibits peripheral nerve degeneration (PND) by targeting Schwann cells in a hydrogen sulfide (H2S)-pathway-dependent manner, but the underlying molecular and pharmacological mechanisms are unclear. We investigated the effect of NEM, an α,ß-unsaturated carboxyl compound, on H2S signaling in in vitro- and ex vivo-dedifferentiated Schwann cells using global proteomics (LC-MS) and transcriptomics (whole-genome and small RNA-sequencing (RNA-seq)) methods. The multi-omics analyses identified several genes and proteins related to oxidative stress, such as Sod1, Gnao1, Stx4, Hmox2, Srxn1, and Edn1. The responses to oxidative stress were transcriptionally regulated by several transcription factors, such as Atf3, Fos, Rela, and Smad2. In a functional enrichment analysis, cell cycle, oxidative stress, and lipid/cholesterol metabolism were enriched, implicating H2S signaling in Schwann cell dedifferentiation, proliferation, and myelination. NEM-induced changes in the H2S signaling pathway affect oxidative stress, lipid metabolism, and the cell cycle in Schwann cells. Therefore, regulation of the H2S signaling pathway by NEM during PND could prevent Schwann cell demyelination, dedifferentiation, and proliferation.