Testing region selection and prognostic analysis of MLH1 promoter methylation in colorectal cancer in China.

Background: MLH1 promoter methylation analysis is recommended in screening for Lynch syndrome (LS) in patients with MLH1-deficient colorectal cancer (CRC). The study aims to identify specific methylation regions in the MLH1 promoter and to evaluate the clinicopathologic characteristics of and prognosis for patients with MLH1 methylation. Methods: A total of 580 CRC cases were included. The DNA mismatch repair (MMR) protein expression was assessed by using immunohistochemistry (IHC). The methylation status of the Regions A, B, C, D, and E in the MLH1 promoter was tested by using bisulfite sequencing PCR. The specificities of the five regions were calculated. Associations between MLH1 methylation and clinicopathologic characteristics were evaluated. Kaplan-Meier analyses for overall survival (OS) were carried out. Results: In 580 CRC cases, the specificities of the methylation test in Regions D and E were both 97.8%. In the MLH1-deficient CRCs, the frequencies of MLH1 methylation and BRAFV600E mutation were 52.6% and 14.6%, respectively; BRAFV600E mutation occurred in 27.7% of patients with MLH1-methylated CRC. In the MMR-deficient patients, compared with MLH1 unmethylation, MLH1 methylation was more common in patients who were aged ≥50 years, female, had no family history of LS-related tumors, and had tumors located at the right colon. In the MMR-deficient patients, the MLH1-methylated cases had lower OS rates than the unmethylated cases with a family history of LS-related tumors (P = 0.047). Conclusions: Regions D and E in the MLH1 promoter are recommended for determining the MLH1 methylation status in screening for LS in MLH1-deficient CRC. In MMR-deficient patients, the MLH1-methylated cases had a worse OS than the unmethylated cases with a family history of LS-related cancer.

Head-to-head comparative study: evaluating three panels for MSI-PCR testing in patients with colorectal and gastric cancer.

AIMS: Due to the lack of large clinical cohorts in the Chinese populations with colorectal cancer (CRC) and gastric cancer (GC), there is no consensus among the preferred panel for microsatellite instability (MSI)-PCR testing. This study aims to evaluate a more appropriate panel. METHODS: We tested the MSI status of 2572 patients with CRC and GC using the NCI panel and 2 mononucleotide panels (5 and 6 mononucleotide panels). Immunohistochemistry (IHC) was employed to perform mismatch repair protein testing in 1976 samples. RESULTS: We collected 2572 patients with CRC and GC. The National Cancer Institute (NCI) panel failed to detect 13 cases. Of the 2559 cases that received results from all three panels, 2544 showed consistent results. In the remaining 15 cases, 9 showed discrepancies between MSI-H and MSI-L, and 6 showed discrepancies between MSI-L and microsatellite stability (MSS). The misdiagnosis rate of MSI-L was significantly lower in two mononucleotide panels than in the NCI panel (12.5% vs 87.5%, p=0.010) in CRC. In patients with GC, only the NCI panel detected three MSI-L cases, while the results of the two mononucleotide panels were one MSI-H and two MSS. Based on their IHC results, the MSI-L misdiagnosis rate of the NCI panel was 33.3%. Furthermore, compared with two mononucleotide panels, the NCI panel had a much lower rate of all loci instability in CRC (90.8% and 90.3% vs 25.2%) and GC (89.5% and 89.5% vs 12.0%). CONCLUSION: In Chinese patients with CRC and GC, the five and six mononucleotide panels have advantages for detecting MSI over the NCI panel.

Isolation of Projection-Specific and Behavior-Relevant Amygdala Circuit for RNA Sequencing.

One of the most sought-after topics in neuroscience is to understand how the environment regulates the activity and function of neural circuitry and subsequently influences relevant behaviors. In response to alterations in the environment, the neural circuits undergo adaptive changes ranging from gene expression changes to altered cellular function. Performing sequencing of the transcriptome involved in these behavior-related circuits will provide clues to accurately dissect the detailed mechanisms of related behavior. Here, we describe methods for marking and collecting the ventral hippocampus-projecting basolateral amygdala neurons, which have been repeatedly implicated in regulation of anxiety-like behavior, and subsequently constructing a library ready for sequencing. Specifically, the reported approaches include adeno-associated virus injection, acute brain slice isolation, cell suspension preparation, cell extraction, and cDNA library construction. By utilizing the techniques described here, researchers can comprehensively investigate the transcriptional levels of neural clusters embedded in particular circuits and discover potential pathogenic and therapeutic targets for behavior-relevant disorders. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Tagging of behavior-related neural circuits Basic Protocol 2: Isolation and capture of fluorescent-positive cells Basic Protocol 3: Foundation of sequencing library.

GABAA(δ) receptor hypofunction in the amygdala-hippocampal circuit underlies stress-induced anxiety.

Dysregulated GABAergic inhibition in the amygdala has long been implicated in stress-related neuropsychiatric disorders. However, the molecular and circuit mechanisms underlying the dysregulation remain elusive. Here, by using a mouse model of chronic social defeat stress (CSDS), we observed that the dysregulation varied drastically across individual projection neurons (PNs) in the basolateral amygdala (BLA), one of the kernel amygdala subregions critical for stress coping. While persistently reducing the extrasynaptic GABAA receptor (GABAAR)-mediated tonic current in the BLA PNs projecting to the ventral hippocampus (BLA â†’ vHPC PNs), CSDS increased the current in those projecting to the anterodorsal bed nucleus of stria terminalis (BLA â†’ adBNST PNs), suggesting projection-based dysregulation of tonic inhibition in BLA PNs by CSDS. Transcriptional and electrophysiological analysis revealed that the opposite CSDS influences were mediated by loss- and gain-of-function of δ-containing GABAARs (GABAA(δ)Rs) in BLA â†’ vHPC and BLA â†’ adBNST PNs, respectively. Importantly, it was the lost inhibition in the former population but not the augmentation in the latter population that correlated with the increased anxiety-like behavior in CSDS mice. Virally mediated maintenance of GABAA(δ)R currents in BLA â†’ vHPC PNs occluded CSDS-induced anxiety-like behavior. These findings clarify the molecular substrate for the dysregulated GABAergic inhibition in amygdala circuits for stress-associated psychopathology.

MADET: a Manually Curated Knowledge Base for Microbiomic Effects on Efficacy and Toxicity of Anticancer Treatments.

A plethora of studies have reported the associations between microbiota and multiple diseases, leading to the development of at least four databases to demonstrate microbiota-disease associations, i.e., gutMDisorder, mBodyMap, Gmrepo, and Amadis. Moreover, gut microbiota mediates drug efficacy and toxicity, whereas a comprehensive database to elucidate the microbiota-drug associations is lacking. Here, we report an open-access knowledge base, MADET (Microbiomics of Anticancer Drug Efficacy and Toxicity), which harbors 483 manually annotated microbiota-drug associations from 26 studies. MADET provides user-friendly functions allowing users to freely browse, search, and download data conveniently from the database. Users can customize their search filters in MADET using different types of keywords, including bacterial name (e.g., Akkermansia muciniphila), anticancer treatment (e.g., anti-PD-1 therapy), and cancer type (e.g., lung cancer) with different types of experimental evidence of microbiota-drug association and causation. We have also enabled user submission to further enrich the data documented in MADET. The MADET database is freely available at https://www.madet.info. We anticipate that MADET will serve as a useful resource for a better understanding of microbiota-drug associations and facilitate the future development of novel biomarkers and live biotherapeutic products for anticancer therapies. IMPORTANCE Human microbiota plays an important role in mediating drug efficacy and toxicity in anticancer treatment. In this work, we developed a comprehensive online database, which documents over 480 microbiota-drug associations manually curated from 26 research articles. Users can conveniently browse, search, and download the data from the database. Search filters can be customized using different types of keywords, including bacterial name (e.g., Akkermansia muciniphila), anticancer treatment (e.g., anti-PD-1 therapy), and cancer type (e.g., lung cancer), with different types of experimental evidence of microbiota-drug association. We anticipate that this database will serve as a convenient platform for facilitating research on microbiota-drug associations, including the development of novel biomarkers for predicting drug outcomes as well as novel live biotherapeutic products for improving the outcomes of anticancer drugs.

Anatomical variations of the branches from left colic artery and middle colic artery at splenic flexure.

BACKGROUND: Variations of the vasculature at splenic flexure by left colic artery (LCA) and middle colic artery (MCA) remain ambiguous. OBJECTIVES: This study aim to investigate the anatomical variations of the branches from LCA and MCA at splenic flexure area. METHODS: Using ultra-thin CT images (0.5-mm thickness), we traced LCA and MCA till their merging site with paracolic marginal arteries through maximum intensity projection (MIP) reconstruction and computed tomography angiography (3D-CTA). RESULTS: A total of 229 cases were retrospectively enrolled. LCA ascending branch approached upwards till the distal third of the transverse colon in 37.6%, reached the splenic flexure in 37.6%, and reached the lower descending colon in 23.1%, and absent in 1.7% of the cases. Areas supplied by MCA left branch and aMCA were 33.2%, 44.5% and 22.3% in the proximal, middle and distal third of transverse colon of the cases, respectively. The accessory MCA separately originated from the superior mesenteric artery was found in 17.9% of the cases. Mutual correlation was found that, when the LCA ascending branch supplied the distal transverse colon, MCA left branch tended to feed the proximal transverse colon; when the LCA ascending branch supplied the lower part of descending colon, MCA left branch was more likely to feed the distal third of transverse colon. CONCLUSIONS: Vasculature at splenic flexure by LCA and MCA varied at specific pattern. This study could add more anatomical details for vessel management in surgeries for left-sided colon cancer.

Raman spectroscopy studies of black phosphorus.

Black phosphorus has important applications in many fields such as optics, optoelectronics and thermals. Many of its excellent properties are related to its special anisotropy. In this work, we adopted Raman spectroscopy, which can obtain fast response optical signals without destroying the structure of the sample, to identify its crystal orientation and explore its thermal and SERS properties. We successfully distinguished the armchair and zigzag directions of black phosphorus by angle-resolved polarized Raman spectroscopy of Ag mode in a less studied orthogonal polarization configuration. Then we used temperature dependent Raman spectroscopy to study its thermal properties. It is found that the first order temperature coefficients of its three Raman vibration modes Ag1, B2g, and Ag2 are -0.0133 cm-1 K-1, -0.0232 cm-1 K-1 and -0.0229 cm-1 K-1, respectively for the 3.2 nm sample. Furthermore, we studied the surface enhancement effect of black phosphorus with different thicknesses as SERS substrates. We found that few-layer black phosphorus has better enhancement effects and its limit of detection for MB and CV are both 10-6M. The analytical enhancement factor of black phosphorus substrates on CV can achieve 1.2 × 103 by calculation. These methods can be extended to other similar two-dimensional materials.

Early life stress induces anxiety-like behavior during adulthood through dysregulation of neuronal plasticity in the basolateral amygdala.

AIMS: Early life stress (ELS) increases the risk of psychiatric diseases such as anxiety disorders and depression in later life. Hyperactivation of the basolateral amygdala (BLA) neurons plays a pivotal role in the pathogenesis of stress-related diseases. However, the functional roles of BLA neurons in ELS-induced anxiety disorders are not completely understood. MAIN METHODS: Mice were subjected to maternal separation (MS) during postnatal days 3 to 21 to mimic ELS. Anxiety-like behavior was tested by open field test (OFT), elevated plus maze (EPM), and novelty suppressed feeding (NSF). Then, c-fos expression, a proxy for neuronal activity, was evaluated by immunofluorescence. Finally, synaptic transmission and intrinsic excitability were measured by whole-cell patch-clamp recordings. KEY FINDINGS: MS significantly increased anxiety-like behavior in adulthood, as indicated by less time spent in the center area of the OFT, less time spent in and fewer entries to the open arms of the EPM, and increased latency to feed in NSF. Mechanistically, MS increased the expression of c-fos in BLA. MS enhanced the excitatory, but not inhibitory, synaptic transmission onto BLA projection neurons (PNs), which was caused by enhanced presynaptic glutamate release. Moreover, MS also markedly increased the intrinsic neuronal excitability of BLA PNs, probably due to the reduced medium afterhyperpolarization (mAHP) in BLA PNs. SIGNIFICANCE: Our results suggest that the changes of neuronal activity and synaptic transmission in the BLA PNs may play a crucial role in ELS-induced anxiety-like behavior, and these findings provide new insights into the pathological mechanisms of stress-related anxiety disorders.

[Analysis of Pollution Characteristics and Sources of Atmospheric VOCs in Ezhou City].

From March 2018 to February 2019, quantitative detection was made of 102 kinds of atmospheric volatile organic compounds (VOCs) using online gas chromatography in Ezhou City. We compared and analyzed the composition, seasonal variation, and diurnal variation of VOCs. Using maximum incremental reactivity (MIR), we estimated the ozone generation potential (OFP) of VOCs. The results show that the annual average volume fraction of atmospheric VOCs in Ezhou is (30.78±15.89)×10-9, and is overall higher in winter than summer, represented by alkane > oxygen > halogenated hydrocarbon > olefin > aromatic hydrocarbon > alkyne. The night volume fraction is higher than in the daytime, and overall the distribution is "double peak". The aromatic hydrocarbons, halogenated hydrocarbons, and OVOCs appear as a "third peak" at 00:00-02:00. Aromatic hydrocarbons and olefins contribute more to the OFP potential of VOCs, with contribution rates of 35.45% and 29.5%, respectively. The highest contribution rate to OFP is ethylene, reaching 24.217%. Analysis of VOC characteristic species found that vehicle exhaust fumes and solvent volatilization are the main sources of VOCs in Ezhou. Of these, motor vehicle emissions are the most important source. Controlling Ezhou's motor vehicle emissions helps to reduce the composition of atmospheric VOCs, thereby reducing ozone production.

Identification of a prefrontal cortex-to-amygdala pathway for chronic stress-induced anxiety.

Dysregulated prefrontal control over amygdala is engaged in the pathogenesis of psychiatric diseases including depression and anxiety disorders. Here we show that, in a rodent anxiety model induced by chronic restraint stress (CRS), the dysregulation occurs in basolateral amygdala projection neurons receiving mono-directional inputs from dorsomedial prefrontal cortex (dmPFC→BLA PNs) rather than those reciprocally connected with dmPFC (dmPFC↔BLA PNs). Specifically, CRS shifts the dmPFC-driven excitatory-inhibitory balance towards excitation in the former, but not latter population. Such specificity is preferential to connections made by dmPFC, caused by enhanced presynaptic glutamate release, and highly correlated with the increased anxiety-like behavior in stressed mice. Importantly, low-frequency optogenetic stimulation of dmPFC afferents in BLA normalizes the enhanced prefrontal glutamate release onto dmPFC→BLA PNs and lastingly attenuates CRS-induced increase of anxiety-like behavior. Our findings thus reveal a target cell-based dysregulation of mPFC-to-amygdala transmission for stress-induced anxiety.

Chronic Stress Oppositely Regulates Tonic Inhibition in Thy1-Expressing and Non-expressing Neurons in Amygdala.

Chronic or prolonged exposure to stress ranks among the most important socioenvironmental factors contributing to the development of neuropsychiatric diseases, a process generally associated with loss of inhibitory tone in amygdala. Recent studies have identified distinct neuronal circuits within the basolateral amygdala (BLA) engaged in different emotional processes. However, the potential circuit involved in stress-induced dysregulation of inhibitory tones in BLA remains elusive. Here, a transgenic mouse model expressing yellow fluorescent protein under control of the Thy1 promoter was used to differentiate subpopulations of projection neurons (PNs) within the BLA. We observed that the tonic inhibition in amygdala neurons expressing and not expressing Thy1 (Thy1+/-) was oppositely regulated by chronic social defeat stress (CSDS). In unstressed control mice, the tonic inhibitory currents were significantly stronger in Thy1- PNs than their Thy1+ counterparts. CSDS markedly reduced the currents in Thy1- projection neurons (PNs), but increased that in Thy1+ ones. By contrast, CSDS failed to affect both the phasic A-type γ-aminobutyric acid receptor (GABAAR) currents and GABABR currents in these two PN populations. Moreover, chronic corticosterone administration was sufficient to mimic the effect of CSDS on the tonic inhibition of Thy1+ and Thy1- PNs. As a consequence, the suppression of tonic GABAAR currents on the excitability of Thy1- PNs was weakened by CSDS, but enhanced in Thy1+ PNs. The differential regulation of chronic stress on the tonic inhibition in Thy1+ and Thy1- neurons may orchestrate cell-specific adaptation of amygdala neurons to chronic stress.

Moderate maternal separation mitigates the altered synaptic transmission and neuronal activation in amygdala by chronic stress in adult mice.

Exposure to moderate level of stress during the perinatal period helps the organisms to cope well with stressful events in their later life, an effect known as stress inoculation. Amygdala is one of the kernel brain regions mediating stress-coping in the brain. However, little is known about whether early life stress may affect amygdala to have its inoculative effect. Here, we observed that moderate maternal separation (MS) from postnatal day 3 to day 21 (D3-21, 1 h per day) significantly alleviated the increased anxiety-like behavior induced by chronic social defeat stress (CSDS) in adulthood, suggesting an obvious inoculative effect of moderate MS. Further studies revealed that MS prevented CSDS-evoked augmentation of glutamatergic transmission onto principal neurons (PNs) in the basolateral amygdala (BLA) by inhibiting presynaptic glutamate release. By contrast, it did not affect GABAergic transmission in BLA PNs, as indicated by unaltered frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs). Moreover, the CSDS-induced increase of neuronal excitability was also mitigated by MS in BLA PNs. In conclusion, our results suggest that MS may have its inoculative effect through alleviating the influences of later life stress on the glutamatergic transmission and neuronal activity in amygdala neurons.

Biotransformation of Resveratrol: New Prenylated trans-Resveratrol Synthesized by Aspergillus sp. SCSIOW2.

Arahypin-16 (1), a new prenylated resveratrol with a unique dihydrobenzofuran ring, has been isolated as a microbial metabolite of resveratrol (2) from whole-cell fermentation of Aspergillus sp. SCSIOW2. The stereochemistry of 1 was determined by ECD calculations. 1 showed about half of the extracellular radical scavenging effect (IC50 = 161.4 µM) compared with resveratrol (IC50 = 80.5 µM), while on biomembranes it exhibited the same range of protection effects against free radicals generated from AAPH (IC50 = 78.6 µM and 87.9 µM).

In silico analysis on structure and DNA binding mode of AtNAC1, a NAC transcription factor from Arabidopsis thaliana.

NAC (NAM/ATAF/CUC) transcription factors regulate the expression of the target genes by formation of NAC-DNA complex, which are involved in development, stress responses and nutrient distribution in many metaphyta plants. AtNAC1, a NAC transcription factor from Arabidopsis thaliana, plays an important role in auxin signaling and root development. In order to understand the structure and DNA binding model of AtNAC1, the 3D structure model of AtNAC1 was constructed and docked with its target DNA. The structure of AtNAC1 monomer contained four α-helices and eight ß-sheets. Two homo monomers of AtNAC1 formed a homo-dimer. The N-terminal sheet S1, Arg24 and Glu31 played an important role in forming AtNAC1 homo-dimer. AtNAC1 dimer interacted with DNA via its core ß-sheet (S5) which contained WKATGKD motif inserting into the major groove of DNA and formed a tight AtNAC1-DNA complex. The DNA sites for AtNAC1 binding were 5'-CTGACGTA-3' and 5'-GATGACGC-3'. Lys102, Ala103, Thr104, Gly105, Lys106, and Asp107 interacting with sugars/bases of DNA were probably responsible for specific recognition of DNA sites. Meanwhile, Arg91, Lys135, and Lys171 binding with phosphate groups of DNA backbone might be the key residues for affinity with DNA. The study provided the in silico framework to understand the interactions of AtNAC1 with DNA at the molecular level.