Cryoprotectants (CPAs) play a critical role in cryopreservation because they can resist the cell damage caused by the freezing process. Current state-of-the-art CPAs are mainly based on an organic solvent dimethyl sulfoxide (DMSO), and several DMSO-cryopreserved cell products have been brought to market. However, the intrinsic toxicity and complex freezing protocol of DMSO still remain as the bottleneck of the wide use for clinical applications. Herein, we reported that betaine, a natural zwitterionic molecule, could serve as a nontoxic and high efficient CPA. At optimum concentration of betaine, different cell types exhibited exceptional post-thaw survival efficiency with ultrarapid freezing protocol, which was straightforward, cost efficient but difficult to succeed using DMSO. Moreover, betaine showed negligible cytotoxicity even after long-term exposure of cells. Mechanistically, we hypothesized that betaine could be ultra-rapidly taken up by cells for intracellular protection during the freezing process. This technology unlocks the possibility of alternating the traditional toxic CPAs and is applicable to a variety of clinical applications.

In this work, to evaluate solute-solute, solute-solvent and phase separation in aqueous systems containing {betaine + poly ethylene glycol dimethyl ether with molar mass 250 g mol-1 (PEGDME250)}, {betaine + K3PO4} and {betaine + K2HPO4}, first water activity measurements were made at 298.15 K and atmospheric pressure using the isopiestic technique. The water iso-activity lines of these three systems were obtained which have positive deviations from the semi-ideal solutions. This suggests that betaine-polymer and betaine-K3PO4 or betaine-K2HPO4 interactions are unfavorable; and these mixtures may form aqueous two-phase systems (ATPSs) at certain concentrations. Indeed the formation of ATPSs was observed experimentally. Then, osmotic coefficient values were calculated using the obtained water activity data; and, using the polynomial method the solute activity coefficients were determined. Using these activity coefficients, the transfer Gibbs energy ([Formula: see text]) values were calculated for the transfer of betaine from aqueous binary to ternary systems consisting polymer (PEGDME250) or salts (K3PO4 and K2HPO4). The obtained positive [Formula: see text] values again indicated that there is unfavorable interaction between betaine and these solutes. Finally, the volumetric and ultrasonic studies were made on these systems to examine the evidence for the nature of interactions between betaine and the studied salts or polymer.

In this work, selenium nanoparticles (Se NPs) stabilized with cocamidopropyl betaine were synthesized for the first time. It was observed that Se NPs synthesized in excess of selenic acid had a negative charge with ζ-potential of -21.86 mV, and in excess of cocamidopropyl betaine-a positive charge with ξ =  + 22.71 mV. The resulting Se NPs with positive and negative charges had a spherical shape with an average size of about 20-30 nm and 40-50 nm, respectively. According to the data of TEM, HAADF-TEM using EDS, IR spectroscopy and quantum chemical modeling, positively charged selenium nanoparticles have a cocamidopropylbetaine shell while the potential- forming layer of negatively charged selenium nanoparticles is formed by SeO32- ions. The influence of various ions on the sol stability of Se NPs showed that SO42- and PO43- ions had an effect on the positive Se NPs, and Ba2+ and Fe3+ ions had an effect on negative Se NPs, which corresponded with the Schulze-Hardy rule. The mechanism of coagulating action of various ions on positive and negative Se NPs was also presented. Also, influence of the active acidity of the medium on the stability of Se NPs solutions was investigated. Positive and negative sols of Se NPs had high levels of stability in the considered range of active acidity of the medium in the range of 1.21-11.98. Stability of synthesized Se NPs stability has been confirmed in real system (liquid soap). An experiment with the addition of Se NPs stabilized with cocamidopropyl betaine to liquid soap showed that the particles of dispersed phases retain their initial distributions, which revealed the stability of synthesized Se NPs.

The present study investigated the effect of foliar application of chitosan at 150 ppm and glycine betaine at 700 ppm on lettuce plants cv. Balady grown under well-watered and water deficit conditions in terms of growth, yield, quality, and water usage efficiency. The study was conducted in Qalubia Governorate, Egypt, during the two seasons of 2020/2021 and 2021/2022 on clay soil. Results indicated that water-stressed plants had a reduction in plant fresh weight, plant height, leaf area, and total yield, chlorophyll content and relative water content, while they exhibited an increase in total soluble solids, nitrate, and proline contents as well as water-use efficiency in both seasons. The foliar application of chitosan or glycine betaine to lettuce significantly improved plant performance under limited and normal irrigation conditions in comparison with untreated plants. The maximum positive effect was for chitosan foliar application. Overall, the results of this study indicated that foliar application of chitosan or glycine betaine was a substitute technology for improving the lettuce yield and quality as well as increasing water use efficiency under both irrigation regimes, but may be more efficient in lettuce plants subjected to a water deficit.

A number of human and animal in vitro or in vivo studies have investigated the relationship between dietary choline and betaine and cancer risk, suggesting that choline and betaine consumption may be protective for cancer. There are also a few epidemiologic studies exploring this relationship, however, with inconsistent conclusions. The PubMed and Embase were searched, from their inception to March 2016, to identify relevant studies and we brought 11 articles into this meta-analysis eventually. The pooled relative risks (RRs) of cancer for the highest versus the lowest range were 0.82 (95% CI, 0.70 to 0.97) for choline consumption only, 0.86 (95%CI, 0.76 to 0.97) for betaine consumption only and 0.60 (95%CI, 0.40 to 0.90) for choline plus betaine consumption, respectively. Significant protective effect of dietary choline and betaine for cancer was observed when stratified by study design, location, cancer type, publication year, sex and quality score of study. An increment of 100 mg/day of choline plus betaine intake helped reduce cancer incidence by 11% (0.89, 95% CI, 0.87 to 0.92) through a dose-response analysis. To conclude, choline and betaine consumption lowers cancer incidence in this meta-analysis, but further studies are warranted to verify the results.

Betaine alleviates high-fat diet-induced fatty liver and prenatal betaine programs offspring hepatic lipid metabolism. Excessive corticosterone (CORT) exposure causes fatty liver in chickens, yet it remains unknown whether and how prenatal betaine modulates the susceptibility of CORT-induced fatty liver later in life. In this study, fertilized eggs were injected with saline or betaine before incubation, and the hatchlings were raised at 8 weeks of age followed by 7 days of subcutaneous CORT injection. CORT-induced fatty liver was less severe in betaine-treated chickens, with significantly reduced oil-red staining and hepatic triglyceride content (P < 0.05). The protective effect of prenatal betaine was associated with significantly up-regulated expression of PPARα and CPT1α, as well as mitochondrial DNA (mtDNA)-encoded genes (P < 0.05). Moreover, betaine rescued CORT-induced alterations in methionine cycle genes, which coincided with modifications of CpG methylation on CPT1α gene promoter and mtDNA D-loop regions. Furthermore, the elevation of hepatic GR protein content after CORT treatment was significantly reduced (P < 0.05), while the reduction of GR binding to the control region of affected genes was significantly increased (P < 0.05), in betaine-treated chickens. These results indicate that in ovo betaine injection protects the juvenile chickens from CORT-induced fatty liver.

Diabetes Mellitus is one of the most worrying issues among illnesses, and its chronic subsequences almost refer to inflammations and infections. The loading and local release of antioxidants to wounds may decrease inflammations. However, the low wettability of PolyPropylene (PP) restricts the drug from loading. So, to increase the adhesion of PP for loading an optimum amount of Betaine Hydrochloride (BET), plasma has been applied in two steps of functionalization and polymerization, which has been confirmed with FE-SEM, ATR-FTIR, and EDX. The new chemistry of the surface led to almost 80% of BET loaded. The drug-releasing ratio studied by HPLC approved the presence of a PEG-like layer, which was coated by polymerization of tetraglyme. To evaluate the wound healing potential of the application of PP meshes treated by plasma, 72 Wistar rats were subdivided into four groups. The skin injury site was removed and underwent biomechanical tests, stereological analysis, and RNA extraction. The results showed a significant improvement in the polymerized scaffold containing BET for skin injury. The present study suggests that the use of a modified PP mesh can induce tissue regeneration and accelerate wound healing at the skin injury site.

Previous research has revealed that the gut microbiome has a marked impact on acute liver failure (ALF). Here, we evaluated the impact of betaine on the gut microbiota composition in an ALF animal model. The potential protective effect of betaine by regulating Toll-like receptor 4 (TLR4) responses was explored as well. Both mouse and cell experiments included normal, model, and betaine groups. The rat small intestinal cell line IEC-18 was used for in vitro experiments. Betaine ameliorated the small intestine tissue and IEC-18 cell damage in the model group by reducing the high expression of TLR4 and MyD88. Furthermore, the intestinal permeability in the model group was improved by enhancing the expression of the (ZO)-1 and occludin tight junction proteins. There were 509 operational taxonomic units (OTUs) that were identified in mouse fecal samples, including 156 core microbiome taxa. Betaine significantly improved the microbial communities, depleted the gut microbiota constituents Coriobacteriaceae, Lachnospiraceae, Enterorhabdus and Coriobacteriales and markedly enriched the taxa Bacteroidaceae, Bacteroides, Parabacteroides and Prevotella in the model group. Betaine effectively improved intestinal injury in ALF by inhibiting the TLR4/MyD88 signaling pathway, improving the intestinal mucosal barrier and maintaining the gut microbiota composition.

Betaine aldehyde dehydrogenase 2 (BADH2) plays a key role in the accumulation of 2-acetyl-1-pyrroline (2AP), a fragrant compound in rice (Oryza sativa). BADH2 catalyses the oxidation of aminoaldehydes to carboxylic acids. An inactive BADH2 is known to promote fragrance in rice. The 3D structure and atomic level protein-ligand interactions are currently unknown. Here, the 3D dimeric structure of BADH2 was modeled using homology modeling. Furthermore, two 0.5 µs simulations were performed to explore the nature of BADH2 dimer structurally and dynamically. Each monomer comprises of 3 domains (substrate-binding, NAD+-binding, and oligomerization domains). The NAD+-binding domain is the most mobile. A scissor-like motion was observed between the monomers. Inside the binding pocket, N162 and E260 are tethered by strong hydrogen bonds to residues in close proximity. In contrast, the catalytic C294 is very mobile and interacts occasionally with N162. The flexibility of the nucleophilic C294 could facilitate the attack of free carbonyl on an aldehyde substrate. Key inter-subunit salt bridges contributing to dimerization were also identified. E487, D491, E492, K498, and K502 were found to form strong salt bridges with charged residues on the adjacent monomer. Specifically, the nearly permanent R430-E487 hydrogen bond (>90%) highlights its key role in dimer association. Structural and dynamic insights of BADH2 obtained here could play a role in the improvement of rice fragrance, which could lead to an enhancement in rice quality and market price.

Cocoamidopropyl betaine (CAPB), which is a biodegradable ampholytic surfactant, has recently been found to dramatically enhance the aerobic digestion of waste activated sludge (WAS) in short-time aerobic digestion (STAD) systems. Therefore, it is important to understand the mechanisms in which CAPB enhances WAS aerobic digestion performance. Results showed that CAPB could dramatically enhance the solubilization of soluble proteins (PN), polysaccharides (PS), nucleic acids (NA) and humic-like substances (HS) in the STAD system within the initial 2 h. Then PN, PS and NA gradually decreased, while HS showed only minor decease. In addition, CAPB increased the proportion of low MW fractions (<20 kDa) from 4.22% to 39.4%, which are more biodegradable. Specific oxygen uptake rates and dehydrogenase enzyme activity results indicated that CAPB markedly improved the aerobic microorganism activities. Microbial community analyses and principle coordinate analyses (PCoA) revealed that CAPB increased the proportion of some functional microorganisms, including Proteobacteria, Planctomycetales, Acinetobacter, Pseudomonas and Aeromonas. The changes driven by CAPB could explain the enhanced performance of the STAD system for WAS aerobic treatment.

Despite epidemiological evidence showing that diets rich in whole grains reduce the risk of chronic life-style related diseases, biological mechanisms for these positive effects are mostly unknown. Increased 5-aminovaleric acid betaine (5-AVAB) levels in plasma and metabolically active tissues such as heart have been associated with consumption of diets rich in whole grains. However, biological effects of 5-AVAB are poorly understood. We evaluated 5-AVAB concentrations in human and mouse heart tissue (3-22 µM and 38-78 µM, respectively) using mass spectrometry. We show that 5-AVAB, at physiological concentration range, dose-dependently inhibits oxygen consumption due to β-oxidation of fatty acids, but does not otherwise compromise mitochondrial respiration, as measured with oxygen consumption rate in cultured mouse primary cardiomyocytes. We also demonstrate that this effect is caused by 5-AVAB induced reduction of cellular L-carnitine. Reduced L-carnitine levels are at least partly mediated by the inhibition of cell membrane carnitine transporter (OCTN2) as evaluated by in silico docking, and by siRNA mediated silencing of OCTN2 in cultured cardiomyocytes. 5-AVAB caused inhibition of β-oxidation of fatty acids is a novel mechanism on how diets rich in whole grains may regulate energy metabolism in the body. Elucidating potentially beneficial effects of 5-AVAB e.g. on cardiac physiology will require further in vivo investigations.

To determine the effects on gingival bleeding, dental biofilm, and salivary flow and pH in patients with gingivitis of using toothpaste with extra-virgin olive oil (EVOO), xylitol, and betaine in comparison to a placebo or commercial toothpaste. This controlled, double blinded, and multicenter randomized clinical trial included patients with gingivitis randomly assigned to one of three groups: test group (EVOO, xylitol, and betaine toothpaste), control group 1 (placebo toothpaste), or control group 2 (commercial toothpaste). Percentage supragingival biofilm and gingival bleeding were evaluated at baseline (T0), 2 months (T2), and 4 months (T4), measuring non-stimulated salivary flow and salivary pH. Comparisons were performed between and within groups. The final study sample comprised 20 in the test group, 21 in control group 1, and 20 in control group 2. In comparison to control group 1, the test group showed significantly greater decreases in gingival bleeding between T4 and T0 (p = 0.02) and in biofilm between T2 and T0 (p = 0.02) and between T4 and T0 (p = 0.01). In the test group, salivary flow significantly increased between T2 and T0 (p = 0.01), while pH alkalization was significantly greater between T4 and T0 versus control group 2 (p = 0.01) and close-to-significantly greater versus control group 1 (p = 0.06). The toothpaste with EVOO, xylitol, and betaine obtained the best outcomes in patients with gingivitis, who showed reductions in gingival bleeding and supragingival biofilm and an increase in pH at 4 months in comparison to a commercial toothpaste.

Although exogenous glycine betaine (GB) and cycloleucine (Cyc) have been reported to affect animal cell metabolism, their effects on plant growth and development have not been studied extensively. Different concentrations of exogenous glycine betaine (20, 40, and 60 mmol L-1) and cycloleucine (10, 20, and 40 mmol L-1), with 0 mmol L-1 as control, were used to investigate the effects of foliar spraying of betaine and cycloleucine on growth, photosynthesis, chlorophyll fluorescence, Calvin cycle pathway, abaxial leaf burr morphology, endogenous hormones, and amino acid content in eggplant. We found that 40 mmol L-1 glycine betaine had the best effect on plant growth and development; it increased the fresh and dry weight of plants, increased the density of abaxial leaf hairs, increased the net photosynthetic rate and Calvin cycle key enzyme activity of leaves, had an elevating effect on chlorophyll fluorescence parameters, increased endogenous indoleacetic acid (IAA) content and decreased abscisic acid (ABA) content, and increased glutamate, serine, aspartate, and phenylalanine contents. However, cycloleucine significantly inhibited plant growth; plant apical dominance disappeared, plant height and dry and fresh weights decreased significantly, the development of abaxial leaf hairs was hindered, the net photosynthetic rate and Calvin cycle key enzyme activities were inhibited, the endogenous hormones IAA and ABA content decreased, and the conversion and utilization of glutamate, arginine, threonine, and glycine were affected. Combined with the experimental results and plant growth phenotypes, 20 mmol L-1 cycloleucine significantly inhibited plant growth. In conclusion, 40 mmol L-1 glycine betaine and 20 mmol L-1 cycloleucine had different regulatory effects on plant growth and development.

Nitric oxide synthase-like activity contributes to the production of nitric oxide in plants, which controls plant responses to stress. This study investigates if changes in ascorbate peroxidase enzymatic activity and glycine betaine content in response to inhibition of nitric oxide synthase-like activity are associated with transcriptional regulation by analyzing transcript levels of genes (betaine aldehyde dehydrogenase) involved in glycine betaine biosynthesis and those encoding antioxidant enzymes (ascorbate peroxidase and catalase) in leaves of maize seedlings treated with an inhibitor of nitric oxide synthase-like activity. In seedlings treated with a nitric oxide synthase inhibitor, transcript levels of betaine aldehyde dehydrogenase were decreased. In plants treated with the nitric oxide synthase inhibitor, the transcript levels of ascorbate peroxidase-encoding genes were down-regulated. We thus conclude that inhibition of nitric oxide synthase-like activity suppresses the expression of ascorbate peroxidase and betaine aldehyde dehydrogenase genes in maize leaves. Furthermore, catalase activity was suppressed in leaves of plants treated with nitric oxide synthase inhibitor; and this corresponded with the suppression of the expression of catalase genes. We further conclude that inhibition of nitric oxide synthase-like activity, which suppresses ascorbate peroxidase and catalase enzymatic activities, results in increased H2O2 content.

The diagnosis and prognosis of chronic kidney disease (CKD) currently relies on very few circulating small molecules, which can vary by factors unrelated to kidney function. In end-stage renal disease (ESRD), these same small molecules are used to determine dialysis dose and dialytic clearance. Therefore, we aimed to identify novel plasma biomarkers to estimate kidney function in CKD and dialytic clearance in ESRD. Untargeted metabolomics was performed on plasma samples from patients with a single kidney, non-dialysis CKD, ESRD and healthy controls. For ESRD patients, pre- and post-dialysis plasma samples were obtained from several dialysis modalities. Metabolomics analysis revealed over 400 significantly different features in non-dialysis CKD and ESRD plasma compared to controls while less than 35 features were significantly altered in patients with a single kidney. N,N,N-trimethyl-L-alanyl-L-proline betaine (TMAP, AUROC = 0.815) and pyrocatechol sulfate (AUROC = 0.888) outperformed creatinine (AUROC = 0.745) in accurately identifying patients with a single kidney. Several metabolites accurately predicted ESRD; however, when comparing pre-and post-hemodialysis, TMAP was the most robust biomarker of dialytic clearance for all modalities (AUROC = 0.993). This study describes TMAP as a novel potential biomarker of kidney function and dialytic clearance across several hemodialysis modalities.

We have previously identified 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) as the most potent substrate-inhibitor of the betaine/GABA transporter 1 (BGT1) (IC50 2.5 µM) reported to date. Herein, we characterize the binding mode of 20 novel analogs and propose the molecular determinants driving BGT1-selectivity. A series of N1-, exocyclic-N-, and C4-substituted analogs was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogs retained subtype-selectivity for hBGT1, though with lower inhibitory activities (mid to high micromolar IC50 values) compared to ATPCA. Further characterization of five of these BGT1-active analogs in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting they interact with the orthosteric site of the transporter. In silico-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 as well as the conformational flexibility of the compounds potentially contribute to the subtype-selectivity of ATPCA and its analogs. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors. The findings may guide the rational design of BGT1-selective pharmacological tool compounds for future drug discovery.

Experimental studies suggest involvement of trimethylamine N-oxide (TMAO) in the aetiology of cardiometabolic diseases and chronic kidney disease (CKD), in part via metabolism of ingested food. Using a comparative biomimetic approach, we have investigated circulating levels of the gut metabolites betaine, choline, and TMAO in human CKD, across animal species as well as during hibernation in two animal species. Betaine, choline, and TMAO levels were associated with renal function in humans and differed significantly across animal species. Free-ranging brown bears showed a distinct regulation pattern with an increase in betaine (422%) and choline (18%) levels during hibernation, but exhibited undetectable levels of TMAO. Free-ranging brown bears had higher betaine, lower choline, and undetectable TMAO levels compared to captive brown bears. Endogenously produced betaine may protect bears and garden dormice during the vulnerable hibernating period. Carnivorous eating habits are linked to TMAO levels in the animal kingdom. Captivity may alter the microbiota and cause a subsequent increase of TMAO production. Since free-ranging bears seems to turn on a metabolic switch that shunts choline to generate betaine instead of TMAO, characterisation and understanding of such an adaptive switch could hold clues for novel treatment options in burden of lifestyle diseases, such as CKD.

Methyltransferases play crucial roles in many cellular processes, and various regulatory mechanisms have evolved to control their activities. For methyltransferases involved in biosynthetic pathways, regulation via feedback inhibition is a commonly employed strategy to prevent excessive accumulation of the pathways' end products. To date, no biosynthetic methyltransferases have been characterized by X-ray crystallography in complex with their corresponding end product. Here, we report the crystal structures of the glycine sarcosine N-methyltransferase from the halophilic archaeon Methanohalophilus portucalensis (MpGSMT), which represents the first structural elucidation of the GSMT methyltransferase family. As the first enzyme in the biosynthetic pathway of the osmoprotectant betaine, MpGSMT catalyzes N-methylation of glycine and sarcosine, and its activity is feedback-inhibited by the end product betaine. A structural analysis revealed that, despite the simultaneous presence of both substrate (sarcosine) and cofactor (S-adenosyl-L-homocysteine; SAH), the enzyme was likely crystallized in an inactive conformation, as additional structural changes are required to complete the active site assembly. Consistent with this interpretation, the bound SAH can be replaced by the methyl donor S-adenosyl-L-methionine without triggering the methylation reaction. Furthermore, the observed conformational state was found to harbor a betaine-binding site, suggesting that betaine may inhibit MpGSMT activity by trapping the enzyme in an inactive form. This work implicates a structural basis by which feedback inhibition of biosynthetic methyltransferases may be achieved.

We have studied here using a number of biophysical tools the effects of osmolytes, betaine, citrulline, proline and sorbitol which differ significantly in terms of their physical characteristics such as, charge distribution, polarity, H-bonding abilities etc, on the fibrillation of insulin. Among these, betaine, citrulline, and proline are very effective in decreasing the extent of fibrillation. Proline also causes a substantial delay in the onset of fibrillation in the concentration range (50-250 mM) whereas such an effect is seen for citrulline only at 250 mM, and in case of betaine this effect is not seen at all in the whole concentration range. The enthalpies of interaction at various stages of fibrillation process have suggested that the preferential exclusion of the osmolyte and its polar interaction with the protein are important in inhibition. The results indicate that the osmolytes are most effective when added prior to the elongation stage of fibrillation. These observations have significant biological implications, since insulin fibrillation is known to cause injection amyloidosis and our data may help in designing lead drug molecules and development of potential therapeutic strategies.

The unicellular alga Dunaliella bardawil is a highly salt-tolerant organism, capable of accumulating glycerol, glycine betaine and β-carotene under salt stress, and has been considered as an excellent model organism to investigate the molecular mechanisms of salt stress responses. In this study, several carotenogenic genes (DbCRTISO, DbZISO, DbLycE and DbChyB), DbBADH genes involved in glycine betaine synthesis and genes encoding probable WRKY transcription factors from D. bardawil were isolated, and promoters of DbCRTISO and DbChyB were cloned. The promoters of DbPSY, DbLycB, DbGGPS, DbCRTISO and DbChyB contained the salt-regulated element (SRE), GT1GMSCAM4, while the DbGGPS promoter has another SRE, DRECRTCOREAT. All promoters of the carotenogenic genes had light-regulated elements and W-box cis-acting elements. Most WRKY transcription factors can bind to the W-box, and play roles in abiotic stress. qRT-PCR analysis showed that salt stress up-regulated both carotenogenic genes and WRKY transcription factors. In contrast, the transcription levels of DbBADH showed minor changes. In D. bardawil, it appears that carotenoid over-accumulation allows for the long-term adaptation to salt stress, while the rapid modulation of glycine betaine biosynthesis provides an initial response.