Alterations in the microbiome correlate with improved metabolism in patients following bariatric surgery. While fecal microbiota transplantation (FMT) from obese patients into germ-free (GF) mice has suggested a significant role of the gut microbiome in metabolic improvements following bariatric surgery, causality remains to be confirmed. Here, we perform paired FMT from the same obese patients (BMI > 40; four patients), pre- and 1 or 6 months post-Roux-en-Y gastric bypass (RYGB) surgery, into Western diet-fed GF mice. Mice colonized by FMT from patients' post-surgery stool exhibit significant changes in microbiota composition and metabolomic profiles and, most importantly, improved insulin sensitivity compared with pre-RYGB FMT mice. Mechanistically, mice harboring the post-RYGB microbiome show increased brown fat mass and activity and exhibit increased energy expenditure. Moreover, improvements in immune homeostasis within the white adipose tissue are also observed. Altogether, these findings point to a direct role for the gut microbiome in mediating improved metabolic health post-RYGB surgery.

Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKβ-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKβ inhibitors, and confirmed by the inability of IKBKB/IKKβ phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKβ’s involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

Short-chain fatty acids (SCFAs) are among the active metabolites in biological process both in the intestinal tract and the bioconversion of organic wastes, which has resulted in various human diseases and environmental problems. In order to accurately detect SCFAs, we introduced a novel extraction sorbent. Electrospun polyacrylonitrile (PAN) nanofiber membrane was synthesized, then poly (3, 4-Ethylenedioxythiophene) (PEDOT) was deposited onto the surface of electrospun PAN nanofibers by in situ polymerization. The morphology of the composite PAN/PEDOT nanofiber was characterized by scanning electronic microscopy (SEM) and FTIR spectrum. PAN/PEDOT was used to isolate and concentrate the SCFAs in waste water and fecal samples before gas chromatography mass spectrometry (GC-MS) analysis. The analytical method was evaluated systematically, and low limits of detection (LODs) of 0.34–0.87 μg/L and good linearity (R² ≥ 0.9953) were obtained. The method was applied successfully for the determination of SCFAs in waste water and fecal samples, with good recovery (87.5–104.6%) and satisfactory reproducibility (relative standard deviation: 6.5–14.1%). The results indicated that the proposed method can be used as a potential approach for the determination of SCFAs with high sensitivity in waste water and biological samples.

Co-occurring symptoms such as depression, anxiety, fatigue, and sleep disorders are frequently comorbid with cancer. The causes of these cancer-related symptom clusters are hypothesized sharing a common biological mechanism. This study explored pattern differences of some gut metabolites (glucocorticoids, short-chain fatty acids, gut microbial metabolites from tryptophan) in plasma samples from patients with four types of cancer. Metabolomics analysis was performed to indicate the differences of metabolites. Discrimination model and diagnostic model were constructed using orthogonal partial least squares discriminant analysis, and differential metabolites were screened, then receiver-operating characteristic curve analysis was performed to evaluate the performance of these models. Melatonin (MLT), indole propionic, and skatole were screened as the common differential metabolites shared by four types of cancer, indicating that the intestinal microbial metabolic pathway of tryptophan plays a key role in the occurrence and development of malignant tumors. The area under the curve values for the potential candidate biomarker predictors in univariate analysis ranged from 0.771 to 0.989, and in multivariate analysis ranged from 0.985 to 1.00. The sensitivity and specificity of the multivariable model were 94.7–100 and 96.4–100%, respectively. These biomarkers also had good performance in discriminating different pairs of cancer. The analysis of gut microbiota metabolites allows us to characterize the common metabolic characteristics of patients with various cancers. The intestinal microbial metabolic pathway of tryptophan plays a key role in the occurrence and development of malignant tumors.

Objective: Our study aimed to compare regional node coverage and doses to the organ at risk (OAR) using conventional technique (CT) vs “AMAROS” (AT) vs intensity-modulated radiation therapy (IMRT) techniques in patients receiving regional nodal irradiation (RNI) for breast cancer (BC). Methods: We included 30 consecutive patients with BC who received RNI including axillary nodes. Two independent and blinded dosimetric RNI plans were generated for all patients. For target volume coverage, we analyzed the V95%, the D95%, the mean and the minimal dose within the nodal station. For hotspots within nodal target volume, we used the V105%, the V108% and the maximal doses. For OAR, lung V20, mean lung and heart doses, the maximal dose to the brachial plexus and the axillary-lateral thoracic vessel junction region were compared between the three techniques. Results: Target volume coverage and hotspots: Mean V95% in stations I, II, III and IV were 35.8% and 75% respectively with CV, 22.59 and 59.9% respectively with AT technique and 45.58 and 99.6% respectively with IMRT with statistically significant differences (p < 0.001). Mean V105% (cc) in axillary and supraclavicular stations were 21.3 and 6.4 respectively with CV, 1.2 and 0.02 respectively with AT technique and 0.5 and 0.4 respectively with IMRT with statistically significant differences (p < 0.001).. OARs: The mean ipsilateral lung V20 was 16.9%, 16.4 and 13.3% with CT, AT and IMRT respectively. The mean heart dose (Gy) was 0.3, 0.2 and 0.2 with CT, AT and IMRT respectively. The maximal dose to the plexus brachial (Gy) was 50.3, 46.3 and 47.3 with CT, AT and IMRT respectively. The maximal dose to the axillary-lateral thoracic vessel junction (Gy) was 52.3, 47.3 and 47.6 with CT, AT and IMRT respectively. The differences were statistically significant for all OAR (p < 0.001). Conclusion: AT is a valuable technique for RNI including axilla in patients with limited sentinel lymph node biopsy involvement without additional axillary lymph node dissection since it decreases hotspots in the target volume and lowers the radiation exposure of the OAR. For more advanced tumors or patients who did not respond to primary systemic therapy, CT or IMRT should be considered because of their better coverage of the potentially residual nodal disease. IMRT combines several advantages of offering high conformal plans, limited hotspots and protection of main OAR. The clinical impact of these dosimetric differences need to be addressed. Advances in knowledge: This study is to our knowledge the first to compare conventional three-dimensional and IMRT techniques for regional nodal irradiation for each nodal station in breast cancer in a context of increasing utilization of axillary irradiation.