[Advances in fermentative production of L-tryptophan: a review].

L-tryptophan is an essential amino acid that is widely used in food, medicine and feed sectors. L-tryptophan can be produced through fermentation, and the main producing strains are engineered Escherichia coli and Corynebacterium glutamicum, which are constructed by rational design methods based on metabolic engineering and synthetic biology. However, due to the long metabolic pathway, complex and unclear regulatory mechanism for L-tryptophan production in microbial cells, the production efficiency and robustness of L-tryptophan producing strains are still low. In this connection, irrational design methods such as laboratory adaptive evolution, are often applied to improve the performance of L-tryptophan producing strains. This review summarizes the recent progress on biosynthesis metabolism of L-tryptophan and its regulation, the construction and optimization of L-tryptophan producing strains, and fermentative production of L-tryptophan, and prospects future development perspective. This review may facilitate research and development for fermentative production of L-tryptophan.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

STUDY QUESTION: Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? SUMMARY ANSWER: NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. WHAT IS KNOWN ALREADY: Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. MAIN RESULTS AND THE ROLE OF CHANCE: DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Gut-Flora-Dependent Metabolite Trimethylamine-N-Oxide Promotes Atherosclerosis-Associated Inflammation Responses by Indirect ROS Stimulation and Signaling Involving AMPK and SIRT1.

Trimethylamine-N-oxide (TMAO), a gut-microbiota-dependent metabolite after ingesting dietary choline, has been identified as a novel risk factor for atherosclerosis through inducing vascular inflammation. However, the underlying molecular mechanism is poorly understood. Using an in vitro vascular cellular model, we found that the TMAO-induced inflammation responses were correlated with an elevation of ROS levels and downregulation of SIRT1 expression in VSMCs and HUVECs. The overexpression of SIRT1 could abrogate both the stimulation of ROS and inflammation. Further studies revealed that AMPK was also suppressed by TMAO and was a mediator upstream of SIRT1. Activation of AMPK by AICAR could reduce TMAO-induced ROS and inflammation. Moreover, the GSH precursor NAC could attenuate TMAO-induced inflammation. In vivo studies with mice models also showed that choline-induced production of TMAO and the associated glycolipid metabolic changes leading to atherosclerosis could be relieved by NAC and a probiotic LP8198. Collectively, the present study revealed an unrecognized mechanistic link between TMAO and atherosclerosis risk, and probiotics ameliorated TMAO-induced atherosclerosis through affecting the gut microbiota. Consistent with previous studies, our data confirmed that TMAO could stimulate inflammation by modulating cellular ROS levels. However, this was not due to direct cytotoxicity but through complex signaling pathways involving AMPK and SIRT1.

Distinct Disruptive Patterns of Default Mode Subnetwork Connectivity Across the Spectrum of Preclinical Alzheimer's Disease.

Background: The early progression continuum of Alzheimer's disease (AD) has been considered to advance through subjective cognitive decline (SCD), non-amnestic mild cognitive impairment (naMCI), and amnestic mild cognitive impairment (aMCI). Altered functional connectivity (FC) in the default mode network (DMN) is regarded as a hallmark of AD. Furthermore, the DMN can be divided into two subnetworks, the anterior and posterior subnetworks. However, little is known about distinct disruptive patterns in the subsystems of the DMN across the preclinical AD spectrum. This study investigated the connectivity patterns of anterior DMN (aDMN) and posterior DMN (pDMN) across the preclinical AD spectrum. Methods: Resting-state functional magnetic resonance imaging (rs-fMRI) was used to investigate the FC in the DMN subnetworks in 20 healthy controls (HC), eight SCD, 11 naMCI, and 28 aMCI patients. Moreover, a correlation analysis was used to examine associations between the altered connectivity of the DMN subnetworks and the neurocognitive performance. Results: Compared to the HC, SCD patients showed increased FC in the bilateral superior frontal gyrus (SFG), naMCI patients showed increased FC in the left inferior parietal lobule (IPL), and aMCI patients showed increased FC in the bilateral IPL in the aDMN; while SCD patients showed decreased FC in the precuneus, naMCI patients showed increased FC in the left middle temporal gyrus (MTG), and aMCI patients also showed increased FC in the right middle frontal gyrus (MFG) in the pDMN. Notably, the FC between the ventromedial prefrontal cortex (vmPFC) and the left MFG and the IPL in the aDMN was associated with episodic memory in the SCD and aMCI groups. Interestingly, the FC between the posterior cingulated cortex (PCC) and several regions in the pDMN was associated with other cognitive functions in the SCD and naMCI groups. Conclusions: This study demonstrates that the three preclinical stages of AD exhibit distinct FC alternations in the DMN subnetworks. Furthermore, the patient group data showed that the altered FC involves cognitive function. These findings can provide novel insights for tailored clinical intervention across the preclinical AD spectrum.