Species-Level Monitoring of Key Bacteria in Fermentation Processes Using Single-Nucleotide Resolved Nucleic Acid Assays Based on CRISPR/Cas12.

Microorganisms can determine the flavor and quality of fermented food, such as Baijiu, which is produced via Daqu fermentation. Therefore, monitoring key microorganisms during fermentation is important for ensuring high-quality fermented food. Here, we report a single-nucleotide resolved nucleic acid assay based on the CRISPR/Cas12 system, enabling the quantification of Bacillus amyloliquefaciens, a key microorganism in Daqu fermentation at the species level. The assay employs an amplification-refractory mutation system derived from PCR to analyze minor genetic differences between different Bacillus species. The utilization of CRISPR/Cas12 further guaranties the specificity of identifying the PCR amplicon and enables the quantification of Bacillus amyloliquefaciens via end-measurement fluorescence. Compared to conventional qPCR, the assay allows for species-level detection of bacteria, thus enabling the precise detection of the Bacillus strain that yields high-level 2,3,5,6-tetramethylpyrazine. The assay promises the precise monitoring of bacterial growth and contribution to flavor during Daqu fermentation, thus facilitating fermented food quality control.

Biochemical metabolism in the anterior cingulate cortex and cognitive function in major depressive disorder with or without insomnia syndrome.

BACKGROUND: Major depressive disorder (MDD) and insomnia have been linked to deficiencies in cognitive performance. However, the underlying mechanism of cognitive impairment in MDD patients with insomnia symptoms (IS) remains unclear. This study aimed to explore the effects of IS in patients with MDD by comparing cognitive function indices among those with IS, those without insomnia symptoms (NIS), and healthy controls (HCs). In addition, we assessed whether the dysfunction of central nervous system (CNS) is one of the important pathophysiologic mechanisms of IS in patients with MDD by comparing the biochemical metabolism ratios in the anterior cingulate cortex (ACC). METHOD: Fifty-five MDD with IS, 39 MDD without IS, and 47 demographically matched HCs underwent the MATRICS Consensus Cognitive Battery (MCCB) assessment and proton magnetic resonance spectroscopy (1H-MRS). MCCB cognitive scores and biochemical metabolism in ACC were assessed and compared between groups. RESULTS: Compared to the HCs group, IS and NIS groups scored significantly lower in seven MCCB cognitive domains (speed of processing, attention/vigilance, working memory, verbal learning, visual learning, reasoning problem solving and social cognition). IS group showed a lower speed of processing and lower Cho/Cr ratio in the left ACC vs. NIS group and HCs. Also, in IS group, the Cho/Cr ratio in the left ACC was positively correlated with the composite T-score. CONCLUSION: Patients with comorbidity of MDD with IS may exhibit more common MCCB cognitive impairments than those without IS, particularly speed of processing. Also, dysfunction of ACC may underlie the neural substrate of cognitive impairment in MDD with IS.

Immediate, non-submerged, three-dimensionally printed, one-piece mandibular molar porous root-analogue titanium implants: A 2-year prospective study involving 18 patients.

This study prospectively evaluated non-submerged, three-dimensionally printed, one-piece molar porous root-analogue titanium implants. A total of 18 non-restorable multiple-rooted teeth in 18 patients, aged 22-64 years, were included in this study. A series of computed tomography images of the mandible were selected and rendered into a digital model. The non-restorable mandibular molars were digitally separated from the surrounding alveolar bone, and served as the template on which the porous root-analogue titanium implants (RAIs) were designed with computer-aided design (CAD) software. The porous molar RAIs were fabricated with the selective laser melting technique (average particle size 20 µm) and inserted into the alveolar sockets after extraction of the non-restorable molars. Definitive restorations were placed after 3 months of uninterrupted healing. Peri-implant clinical and radiographic measurements were obtained 2 years later. All patients functioned well following 2 years of functional loading, and peri-implant clinical and radiographic measurements demonstrated implant stability. No implants were lost at the 2-year follow-up, and the survival rate was 100%. Three-dimensionally printed one-piece molar porous RAIs may be a promising option for the replacement of non-restorable molars that are planned for extraction. Additional studies are required to evaluate the long-term survival of implants fabricated using this technique.

Label-Free Detection of Transgenic Crops Using an Isothermal Amplification Reporting CRISPR/Cas12 Assay.

Current tools for detecting transgenic crops, such as polymerase chain reaction (PCR), require professional equipment and complex operation. Herein, we introduce a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to analyze transgenes by designing an isothermal amplification to serve as the amplified reporter, allowing an isothermal and label-free detection of transgenic crops. The use of Cas12a allowed direct and specific recognition of transgenes. To enhance the sensitivity of the assay, we used rolling circle amplification (RCA) to monitor the recognition of transgenes by designing the RCA primer as the cleavage substrate of Cas12a. The presence of transgenes can be detected by monitoring the G-quadruplex in RCA amplicon using a G-quadruplex binding dye, N-methyl mesoporphyrin IX (NMM). We termed the assay as isoCRISPR and showed that the assay allowed distinguishing transgenic corn cultivars ("Bt11" and "MON89034") from nontransgenic corn cultivars ("yellow", "shenyu", "xianyu", and "jingke"). The isoCRISPR assay will enrich the toolbox for transgenic crop identification and broaden the application of CRISPR/Cas in food authenticity and safety.

Amplification Refractory Mutation System (ARMS)-PCR for Waxy Sorghum Authentication with Single-Nucleotide Resolution.

Waxy sorghum has greater economic value than wild sorghum in relation to their use in food processing and the brewing industry. Thus, the authentication of the waxy sorghum species is an important issue. Herein, a rapid and sensitive Authentication Amplification Refractory Mutation System-PCR (aARMS-PCR) method was employed to identify sorghum species via its ability to resolve single-nucleotide in genes. As a proof of concept, we chose a species of waxy sorghum containing the wxc mutation which is abundantly used in liquor brewing. The aARMS-PCR can distinguish non-wxc sorghum from wxc sorghum to guarantee identification of specific waxy sorghum species. It allowed to detect as low as 1% non-wxc sorghum in sorghum mixtures, which ar one of the most sensitive tools for food authentication. Due to its ability for resolving genes with single-nucleotide resolution and high sensitivity, aARMS-PCR may have wider applicability in monitoring food adulteration, offering a rapid food authenticity verification in the control of adulteration.

Correlations Between Working Memory Impairment and Neurometabolites of the Prefrontal Cortex in Drug-Naive Obsessive-Compulsive Disorder.

PURPOSE: This study aimed to investigate the mechanism of working memory (WM) impairment in drug-naive obsessive-compulsive disorder (OCD) by using neuropsychological tests and proton magnetic resonance spectroscopy (1H-MRS). PATIENTS AND METHODS: A total of 55 patients with drug-naive OCD and 55 healthy controls (HCs) were recruited for this study. The working memory (WM) was evaluated using the digit span test (DST), visual space memory test (VSMT), and the 2-back task and stroop color word test (SCWT). The bilateral metabolite levels of the prefrontal cortex (PFC) were evaluated by 1H-MRS, then determined the ratios of N-acetyl aspartate (NAA), choline-containing compounds (Cho), and myo-inositol (MI) to creatine (Cr). The independent sample t-test was used to analyse the differences in WM performance and neurometabolite ratios. Multivariate linear regression analysis was performed to screen the influential factors of WM, with an introduction level of 0.05 and a rejection level of 0.10. RESULTS: 1) Patients with OCD performed significantly worse on DST (score), VSMT (score), 2-back task (accuracy rate), SCWT (execution time) when compared with HCs. 2) NAA/Cr and Cho/Cr in the left PFC (lPFC) and MI/Cr ratios in the bilateral PFC of OCD patients were significantly lower when compared to HCs. 3) For OCD patients, the NAA/Cr ratio in the lPFC was negatively correlated with the score of DST (forwards), the Cho/Cr ratio in the lPFC was positively correlated with the accuracy rate of 2-back task, and the MI/Cr ratio in the right PFC (rPFC) was positively correlated with the score of DST (forwards) and the accuracy rate of VSMT. We also found that the compulsive symptoms showed a positive correlation with MI/Cr ratio of the rPFC. CONCLUSION: Drug-naive OCD patients have demonstrated WM impairments, including phonological loop, visual-spatial sketchpad and central executive system, and the WM impairments might be associated with hypometabolism in the PFC, especially the lPFC.

Dynamic changes of volatile compounds and bacterial diversity during fourth to seventh rounds of Chinese soy sauce aroma liquor.

Chinese soy sauce aroma liquor (CSSL) is a famous Baijiu. Multiple rounds of fermentation, the characteristic of CSSL processing, contributes to the differences in the quality of the liquor of different rounds. In this study, the grains on cooled, stacked, and fermented stages of 4th to 7th rounds were taken, of which the environmental factors, bacterial diversity, and volatile compounds were comprehensively analyzed. Lactobacillaceae, Bacillaceae, Thermoactinomycetaceae, and Enterobacteriaceae were the top four families, of which Lactobacillaceae dominated the fermented stage of each round. Principal component analysis (PCA) and principal coordinate analysis (PCoA) supported the popular view that the liquors of 3rd to 5th rounds possess the best quality. Lactobacillaceae is an extremely critical bacterium for CSSL fermentation. This study provides comprehensive understanding regarding the dynamic changes in fermented grains during the 4th to 7th rounds, which could help to improve the processing technology of CSSL.

The influences of behavioral and psychological interventions on the neurological function and MMSE scores in Parkinson's disease patients.

OBJECTIVE: To analyze the effects of behavioral and psychological interventions on the neurological function in Parkinson's disease (PD) patients. METHODS: A total of 103 patients with PD admitted to our hospital were randomly divided into the observation group (OG, n=52) or the control group (CNG, n=51). The CNG was treated with routine nursing, while the OG was treated with behavioral and psychological interventions. RESULTS: At 1, 2, and 3 months after the nursing, the National Institutes of Health Stroke Scale (NIHSS) scores, the unified PD disease rating scale III (UPDRS-III) scores, and the Pittsburgh Sleep Quality Index (PSQI) scores in both groups were lower than they were before the nursing, the NIHSS, UPDRS-III, and PSQI scores in the OG were lower than they were in the CNG, the General Self-efficacy Scale (GSES) and Mini-Mental State Examination (MMSE) scores and the Barthel scores in both groups were higher than they were before the nursing, and the GSES, MMSE, and Barthel scores in the OG were higher than they were in the CNG (P < 0.05). CONCLUSION: Behavioral and psychological interventions can improve PD patients' impaired neurological function, self-efficacy, cognitive function, motor dysfunction, sleep quality, and quality of life.

Label-free DNAzyme assays for dually amplified and one-pot detection of lead pollution.

Lead pollution in water and soil often transfers to food, advocating tools for on-site detection of lead pollution to ensure both environmental and food safety. We proposed a label-free, dually amplified and homogeneous DNAzyme assay for sensitive and one-pot detection of lead pollution. Instead of using chemically modified DNA substrate, a structure-response digestion process was introduced to monitor Pb2+ presence-induced cleavage process of unlabeled substrate, further amplifying the response signals and eliminating the use of labeled DNA probes. The DNAzyme assay allowed to detect Pb2+ as low as 0.12 nM and endued a dynamic range from 0.1 nM to 30 nM. In addition, it can specifically identify Pb2+ among other metal ions. We demonstrated that the DNAzyme assay can precisely detect Pb2+ in tap water, milk and fish. Thus, the DNAzyme assay is promising for on-site monitoring lead pollution risk and ensuring environmental and food safety.

Directly profiling intact Staphylococcus aureus in water and foods via enzymatic cleavage aptasensor.

Staphylococcus aureus (S. aureus) causes serious food-borne diseases, and tools able to directly profile intact S. aureus would greatly facilitate food safety and public health. Herein, we proposed a biosensing platform for culture-independent and separation-free profiling S. aureus, thus allow us to directly detect intact S. aureus in complex samples. The binding protection effect of aptamer-cell complex was introduced to construct the aptasensor, and it allowed to eliminate the optimization of aptamer probe sequences. The proposed aptasensor, terms enzymatic cleavage aptasensor could achieve a sensitive (a detection limit of 64 CFU/mL) and broad-concentration quantification (dynamic range 102-107 CFU/mL) of S. aureus. Furthermore, it could specifically identify intact S. aureus in complex samples, and the quantifying of S. aureus was achieved in tap water, milk and porker with high precision. Therefore, enzymatic cleavage aptasensor could be a good candidate for on-site biosensing platform of S. aureus, as well as other pathogens by replacing the aptamer sequences.

Investigating the Variation of Volatile Compound Composition in Maotai-Flavoured Liquor During Its Multiple Fermentation Steps Using Statistical Methods.

The use of multiple fermentations is one of the most specific characteristics of Maotai--flavoured liquor production. In this research, the variation of volatile composition of Maotai-flavoured liquor during its multiple fermentations is investigated using statistical approaches. Cluster analysis shows that the obtained samples are grouped mainly according to the fermentation steps rather than the distillery they originate from, and the samples from the first two fermentation steps show the greatest difference, suggesting that multiple fermentation and distillation steps result in the end in similar volatile composition of the liquor. Back-propagation neural network (BNN) models were developed that satisfactorily predict the number of fermentation steps and the organoleptic evaluation scores of liquor samples from their volatile compositions. Mean impact value (MIV) analysis shows that ethyl lactate, furfural and some high-boiling-point acids play important roles, while pyrazine contributes much less to the improvement of the flavour and taste of Maotai-flavoured liquor during its production. This study contributes to further understanding of the mechanisms of Maotai-flavoured liquor production.

Characterization and comparison of the pungent components in commercial Zanthoxylum bungeanum oil and Zanthoxylum schinifolium oil.

In the present study, the characterization and comparison of the pungent components in commercial Z. bungeanum oils and Z. schinifolium oils were investigated. By high-performance liquid chromatography (HPLC)-mass spectrometry (MS/MS) analysis, the major alkylamides in Z. bungeanum and Z. schinifolium oils were identified as hydroxy-ε-sanshool, hydroxy-α-sanshool, hydroxy-ß-sanshool, hydroxy-γ-sanshool, hydroxy-γ-isosanshool, bungeanool, isobungeanool, and tetrahydrobungeanool, respectively. Hydroxy-α-sanshool was found to be the most abundant alkylamide in all oils. The levels of hydroxy-ε-sanshool and hydroxy-ß-sanshool in Z. bungeanum oils were comparable to that in Z. schinifolium oils, whereas Z. bungeanum oils contained significantly (P less than 0.05) higher levels of hydroxy-γ-isosanshool, bungeanool, isobungeanool, and tetrahydrobungeanool, compared with Z. schinifolium oils. Furthermore, principal component analysis (PCA) indicated that Z. bungeanum oil and Z. schinifolium oil were clearly classified by HPLC fingerprinting profiles and concentrations of alkylamides. In addition, the results of PCA suggested that alkylamides, such as hydroxy-γ-sanshool and bungeanool, could be potential markers to distinguish Z. bungeanum oil and Z. schinifolium oil. The results from this study could be used to discriminate the different flavor characterization and control the quality of commercial Z. bungeanum oil and Z. schinifolium oil.

Detection of organophosphorus pesticides using potentiometric enzymatic membrane biosensor based on methylcellulose immobilization.

In this research, a new potentiometric enzymatic membrane biosensor for the detection of organophosphorus pesticides (OPs) was constructed. The basic element of this biosensor was a pH electrode modified with an immobilized acetylcholinesterase layer formed by entrapment with methylcellulose, N,N-dimethylformamide, and bovine serum albumin. The response characteristics of the biosensor were measured and discussed. It was shown that there is a good linear relationship between the inhibition rate and the negative logarithm of OPs concentration in the range from 10(-7) to 10(-5) mol/L, with the detection limits of 10(-7) mol/L for the five pesticides. Moreover, the biosensor could resist the disturbances of below 10(-6) mol/L of Cu(2+) and Pb(2+), and below 10(-5) mol/L of Cd(2+). In addition, the measurement results obtained by the biosensor method were in good agreement with those obtained by the gas chromatography method. This method was successfully applied to detect OPs that remained in the potted lettuce.