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In the present work, a novel, nutritious food product, a spinach smoothie, was developed. Due to microbial spoilage and quality degradation, the smoothie's limited shelf life was the main issue. For this purpose, natural preservatives (garlic filtrate, ginger filtrate, and a mixture of garlic + ginger filtrate) in comparison to synthetic preservatives (sodium benzoate, citric acid, and a combination of sodium benzoate + citric acid) were added to enhance the shelf life. Further, physicochemical analyses such as pH, specific gravity, antioxidant potential, mineral content, microbial content, and organoleptic properties were evaluated during storage of 28 days. Maximum antioxidant activities for DPPH (41.22 - 32.27%), FRAP (33.21 - 29.35%), TPC (27.11 - 20.84 mg/mL GAE), and vitamin C (31.65 - 27.37 mg/100 mL) were observed with garlic + ginger filtrate smoothie from initial to final day of storage. The highest total bacterial count was observed in the control sample of 30 × 10³ - 530 × 10³ CFU/mL, and the lowest count was seen in garlic-ginger filtrate smoothie of 12 × 10³ - 20 × 10³ CFU/mL during the 28-day storage. The maximum score for sensorial acceptability was observed with garlic + ginger filtrate. Therefore, biopreservatives such as garlic + ginger filtrate at 1% had the highest efficacy level, and were economically suitable. Thus, combining natural preservatives like garlic and ginger with additional nutritional benefits could replace synthetic preservatives. [ABSTRACT FROM AUTHOR]

Present study was conducted to investigate the content of toxic heavy metals; lead, cadmium, manganese, nickel and zinc in organs of edible fish of River Ravi. Samples of Labeo rohita, Cyprinus carpio, Gibelion catla and Cirrhinus mrigala were captured from two sites at River Ravi i.e. Kala Khatai and Head Balloki. Zinc and lead content was higher in organs of fish specimens collected from Head Balloki than those collected from Village Kala Khatai. Higher average cadmium content was detected in most of the organs of fish collected from Head Balloki than those of Village Kala Khatai. However, higher average manganese content was found in examined organs of fish captured from Village Kala Khatai than those of Head Balloki. Lead and cadmium content in muscles of fish collected from Head Balloki was higher than acceptable level proposed by European Commission (0.2 µg/g and 0.05 µg/g, respectively). Hepatosomatic index of fish species (except L. rohita) collected from Head Balloki was lower than those of Village Kala Khatai. Study has led to the conclusion that edible fish of River Ravi is contaminated with toxic metals and contamination level is higher in fish at Head Balloki as compared to that observed at Village Kala Khatai. [ABSTRACT FROM AUTHOR]

The light plays a key role in regulating anthocyanin biosynthesis in plants. However, the molecular basis of anthocyanin synthesis in grape skins irradiated at night with supplemental white light (W), red light (R), blue light (B), and blue light for 3 h, 6 h, 9 h, and 12 h (B3, B6, B9, and B12) is not known. In the present study, the total anthocyanin content in grape skins was significant under different light (DL) and blue-light time (DT) treatments, and the best result was obtained with B9. Analysis of RNA-seq data determined that the key genes in the anthocyanin synthesis pathway, PAL, CHS, DFR, GT, CFIP, VIT_14s0068g00920, VIT_06s0009g02860, and VIT_06s0004g08150, were significantly up-regulated under night-time supplemental light treatments, which led to the significant expression of the phenylpropanoid biosynthesis, phenylalanine metabolism, flavonoid biosynthesis, flavone and flavonol biosynthesis, and the anthocyanin biosynthesis pathway, which were significantly expressed, promoting the accumulation of anthocyanin. The B caused significant expression of PAL (VIT_08s0040g01710), CFIP (VIT_13s0067g03820, VIT_13s0067g02870) and DFR (VIT_18s0001g12800), which may be one of the reasons for the better effect of B compared to W anthocyanin enrichment in grape skins. The DT treatment resulted in significant expression of GT (VIT_11s0052g01600), Peroxidase, VIT_09s0018g01190, and VIT_11s0037g00570. In addition, many TFs such as bHLH, MYB, ERF, WRKY, C2H2, MYB-related, and NAC were found to be involved in the synthesis of anthocyanins under light regulation. These results provide new insights into plants’ nocturnal supplemental-light regulation of anthocyanin accumulation.

Present study was based on determination of soil quality to assess its suitability for aquaculture activities from thirty-one selected areas situated in four division of Punjab. Results indicated that soil particle size distribution was suitable for construction of fish ponds at most of the sites in Lahore and Sahiwal. In Gujranwala, about 50% of soil samples contained higher sand content (> 60%). Clay particles were found to be less than 25% in 75% of the soil samples whereas sand content was higher than 50% in 25% of the samples collected from Sargodha division. Higher soil pH (> 7.0) found at all sites showed calcareous nature of soil and indicated that liming soil to increase pH was not a necessity in these areas. Soil at all the studied areas of Sargodha was found to be saline or very strongly saline except at Village Hernali in Mianwali district. It was found that about 83%, 100% and 80% of sites studied in Lahore, Sahiwal and Sargodha can be used for fish culture respectively. However, none of the site in Gujranwala was found to be suitable for aquaculture if used in present form. Appropriate soil management techniques can be adopted to use the land with unsuitable soil particle size distribution for aquaculture. Saline nature of soil in Sargodha division must be considered while selecting cultureable species of fish/shrimps in these areas. [ABSTRACT FROM AUTHOR]

The study was designed to assess the effects of periphyton grown on different substrates on water quality and production of major carps cultured in zero water exchange system. Three experimental treatments included; T1 (Control): without substrate, T2: using PVC pipes substrates, T3, using Bamboo poles substrates. Fish seed of Labeo rohita: Cirrhinus mrigala and Gibelion catla were stocked in cemented tanks in 1:1:1 ratio at stocking density of 3 fishm-2 for 60 days period. Survival rate of L. rohita, C. mrigala and G. catla was significantly higher (p<0.05) in substrates based treatments. In T1, T2 & T3, it was found to be 34.09%, 81.82% & 90.91% for L. rohita; 22.73%, 100.0% & 100.0% for C. mrigala and 36.36%, 90.91% & 100.0% for G. catla respectively. Total fish biomass of L. rohita, C. mrigala and G. catla harvested at the end of trial was 2.24, 4.32 and 1.42 times higher in treatment with bamboo poles as substrates and 1.18, 3.64 and 0.75 time higher in PVC pipes based treatment than that of control. Concentration of ammonia and nitrite was found to be significantly low in treatments with periphyton than control. High fish survival in periphyton based systems can be attributed to bioremediation through bioprocesses of their ubiquitous microbial biofilms. [ABSTRACT FROM AUTHOR]

Previous results indicated that the flavonoid profiles might have varietal differences in pomelo, but detailed information is unknown. We previously isolated 4 new flavonoids, cigranoside C, D, E, F, in Citrus grandis Shatianyu pulp. However, their distribution in different pomelo cultivars remains to be explored. Therefore, the flavonoid profiles and in vitro bioactivity of the pulp from 5 pomelo and 1 grapefruit cultivars commonly consumed in China were investigated. Fourteen flavonoids were identified, cigranoside C, D, E were detected in these pomelo and grapefruit. Naringin and cigranoside C were the major flavonoids in grapefruit, Guanximiyu-W, Guanximiyu-R and Liangpingyu, while melitidin and rhoifolin was the predominant flavonoid in Shatianyu and Yuhuanyu, respectively. Pomelo and grapefruit showed strong antioxidant activity, and were potent inhibitors of pancreatic lipase with IC50 values of 11.4–72.6 mg fruit/mL except Shatianyu. Thus, pomelo and grapefruit are natural antioxidants and possess anti-obesity potential.

Objective: Human osteoarthritic (OA) cartilage type-II collagen is preferentially cleaved by the proinflammatory cytokine-induced matrix metalloproteinases-13 (MMP-13). Interferon-gamma (IFN-gamma) potently inhibits interleukin-1 (IL-1)-induced MMP-13 expression in healthy chondrocytes. Our goal was to study the previously unknown impact of IFN-gamma on MMP-13 in OA and compare the levels and functional activity of IFN-gamma receptor (IFN-gammaR1) in healthy and OA chondrocytes.Methods: Chondrocytes were obtained from OA patients and non-arthritic control subjects and treated with IL-1+ or- IFN-gamma. MMP-13 mRNA and protein expression were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. IFN-gammaR1 expression was assessed by flow cytometry, immunoprecipitation and immunohistochemistry with fluorescein-labeled antibody. IFN-gammaR1 was neutralized with its antibody and signal transducer and activator of transcription 1 (STAT1) phosphorylation analyzed by Western blotting. OA chondrocytes were also transfected with control and IFN-gammaR1 expression vectors.Results: OA chondrocytes displayed a drastically impaired MMP-13 suppression by IFN-gamma compared to control cells. IFN-gammaR1 levels were significantly decreased in OA chondrocytes as assessed by flow cytometry, immunoprecipitation and immunohistochemistry. Consequently, IFN-gamma-stimulated STAT1 phosphorylation mediated by IFN-gammaR1 was also considerably reduced in OA patient chondrocytes. IFN-gammaR1 overexpression in OA cells restored MMP-13 suppression by IFN-gamma.Conclusions: Ability of IFN-gamma to suppress IL-1-induced MMP-13 expression is diminished in OA chondrocytes due to decreased IFN-gammaR1 levels, activity and impaired downstream signal transduction. Therefore, IFN-gammaR1 modulation and weakened endogenous IFN-gamma response may be important mechanisms in OA pathogenesis and cartilage degradation. [ABSTRACT FROM AUTHOR]

Lead (Pb) is a pestilent and relatively nonbiodegradable heavy metal, which causes severe health effects by inducing inflammation and oxidative stress in animal and human tissues. This is because of its significant tolerance and capability to bind Pb (430 mg/L) and thermodynamic fitness to sequester Pb in the Freundlich model (R2 = 0.98421) in vitro. Lactobacillus acidophilus KLDS1.1003 was selected for further in vivo study both in free and maize resistant starch (MRS)–based microencapsulated forms to assess its bioremediation aptitude against chronic Pb lethality using adult female BALB/c mice as a model animal. Orally administered free and microencapsulated KLDS 1.1003 provided significant protection by reducing Pb levels in the blood (127.92 ± 5.220 and 101.47 ± 4.142 µg/L), kidneys (19.86 ± 0.810 and 18.02 ± 0.735 µg/g), and liver (7.27 ± 0.296 and 6.42 ± 0.262 µg/g). MRS-microencapsulated KLDS 1.0344 improved the antioxidant index and inhibited changes in blood and serum enzyme concentrations and relieved the Pb-induced renal and hepatic pathological damages. SEM and EDS microscopy showed that the Pb covered the surfaces of cells and was chiefly bound due to the involvement of the carbon and oxygen elements. Similarly, FTIR showed that the amino, amide, phosphoryl, carboxyl, and hydroxyl functional groups of bacteria and MRS were mainly involved in Pb biosorption. Based on these findings, free and microencapsulated L. acidophilus KLDS 1.0344 could be considered a potential dietetic stratagem in alleviating chronic Pb toxicity.

Puerarin is a bioactive flavonoid isolated from Kudzu roots that possesses numerous health benefits. However, its poor bioavailability and existing complex delivery systems with safety issues are challenging tasks for its incorporation into functional foods. Preparing modified-starch-stabilized Pickering emulsions containing microencapsulated puerarin with improved bioaccessibility was the key objective of the present research work. Acid-hydrolyzed high-amylose Pueraria montana starch (PMS) was modified with octenyl succinic anhydride (OSA) and evaluated as an emulsifier to prepare emulsions. The FTIR, SEM, and XRD results showed that PMS was successfully modified. Furthermore, the emulsification index (EI), mean droplet size, and ζ-potential values showed that modified starch with a higher degree of substitution (DS) enhanced the storage stability of emulsions. Similarly, the retention degree and encapsulation efficiency results of puerarin proved the assumption after storage of 16 d. The Pickering emulsions also helped in the controlled release of microencapsulated puerarin in vitro. The study outcomes proved that Pickering emulsions stabilized with OSA-modified PMS have promising applicability in functional foods as efficient food-grade delivery systems, enhancing oral supplementation and accessibility of puerarin.

Purpose – Internal organizational orientation of service quality and its impact on service delivery performance of the employees have received considerable attention from financial management literature. The purpose of this paper is to address this issue by conducting empirical research focusing on the Pakistani Islamic banking industry. It conceptualizes and measures key determinants of internal organizational orientation of service quality from the employees' perspective. Design/methodology/approach – The data were collected from a sample of 150 employees of pure Islamic banks and conventional banks with IBBs (Islamic Banking Branches or windows) across the entire country. The paper uses principal component factor analysis and regression methods. Findings – Statistical results demonstrate that the employee perceptions of organizational service quality orientation mainly depends upon four main predictors: employees' perception about training and development; development and positioning of Islamic banking products/service concept; customer service orientation; and employees' service quality performance. Principal component factor analysis results indicate four predictive internal organizational service quality orientation factors (ISQF) where 16 per cent of the variation is being explained by employee perception of organizational orientation towards employees' training and development (ISQF1), 13 per cent variation explained employee perception of organizational orientation towards development and positioning of Islamic banking products/service concept (ISQF2), 11 per cent variation explained by employee perception of organizational service quality orientation towards customer service orientation (ISQF3), and 10 per cent variation explained employee perception of organizational service quality orientation towards employees' service quality performance (ISQF4). Originality/value – Management of Islamic Banks in Pakistan need to be mindful about the fact that ISQFs identified by this study have the potential to indirectly influence customer perceptions through effective employees' recruitment and selection criteria, complemented by training to improve service oriented skills and knowledge development about Sh´ria principles related with the products/services offered by Islamic banks in Pakistan. [ABSTRACT FROM AUTHOR]

Lead (Pb) is a well-recognized and potent heavy metal with non-biodegradable nature and can induce the oxidative stress, degenerative damages in tissues, and neural disorders. Certain lactic acid bacterial strains retain the potential to mitigate the lethal effects of Pb. The present work was carried out to assess the Pb bio-sorption and tolerance capabilities of Lactobacillus plantarum spp. Furthermore, potato resistant starch (PRS)-based microencapsulated and non-encapsulated L. plantarum KLDS 1.0344 was utilized for bioremediation against induced chronic Pb toxicity in mice. The experimental mice were divided into two main groups (Pb exposed and non-Pb exposed) and, each group was subsequently divided into three sub groups. The Pb exposed group was exposed to 100 mg/L Pb(NO3)2 via drinking water, and non-Pb exposed group was supplied with plain drinking water during 7 weeks prolonged in vivo study. The accumulation of Pb in blood, feces, renal, and hepatic tissues and its pathological damages were analyzed. The effect of Pb toxicity on the antioxidant enzyme capabilities in blood, serum, as well as, on levels of essential elements in tissues was also calculated. Moreover, KLDS 1.0344 displayed remarkable Pb binding capacity 72.34% and Pb tolerance (680 mg/L). Oral administration of both non- and PRS- encapsulated KLDS 1.0344 significantly provided protection against induced chronic Pb toxicity by increasing fecal Pb levels (445.65 ± 22.28 μg/g) and decreasing Pb in the blood up to 137.63 ± 2.43 μg/L, respectively. KLDS 1.0344 microencapsulated with PRS also relieved the renal and hepatic pathological damages and improved the antioxidant index by inhibiting changes in concentrations of glutathione peroxidase, glutathione, superoxide dismutase, malondialdehyde, and activated oxygen species, which were affected by the Pb exposure. Overall, our results suggested that L. plantarum KLDS 1.0344 either in free or encapsulated forms hold the potentiality to deliver a dietetic stratagem against Pb lethality.

This study investigated the effects of heat treatment after purification on dissociation, aggregation, and structural modification of polyphenol oxidase (PPO) activity from apple (Malus domestica) juice. PPO activity at the 70°C for 10 min was still activated and drastically decreased since 20–60 min with catechol and pyrogallol as substrate. Moreover, spectral results of fluorescence and circular dichroism (CD) indicated that increasing temperature for shorter and longer durations can cause reorganization of the secondary structure of PPO and demolished the native configuration of PPO respectively. Compared with native PPO, all thermally treated PPO showed reduced activity with gradually increasing particle size shift toward section III of some fully assembled proteins treated at 70°C for 10 min (2,670 nm). Polyacrylamide gel electrophoresis (PAGE) analysis also exhibited the increase in protein content at the 70°C for 10 min with molecular size 35 kDa (7.7 ± 0.016c). Hence, thermally treated juice subjected to purification at high temperature for a short time could induce the aggregation of protein and is not really effective for PPO inactivation. For PPO, higher degree of long duration can induce the inactivation of the enzyme after processing.

The biosynthesis of penicillin G (PG) is compartmentalized, which forces penicillin and its intermediates to cross the membrane barriers. Although many aspects around the penicillin intermediates traffic system remain unclosed, the transmembrane transporter protein involvement has been only predicted. In the present work, detection of PG and isopenicillin N (IPN) in Monascus ruber M7 was performed and functions of mfst gene as a transporter were investigated by the combination of gene deletion (Δmfst) complementation (ΔmfsT::mfsT) and overexpression (M7::PtrpC-mfsT). While, the feeding of PG pathway precursor side chain and amino acids, i.e., phenylacetic acid, D-valine, and L-cysteine was performed for the interpretation of mfsT gene role as an intermediate transporter. The results showed that, the feeding of phenylacetic acid, D-valine, and L-cysteine possessed a significant effect on morphologies, secondary metabolites (SMs) production of all above-mentioned strains including M. ruber M7. The results of UPLC-MS/MS revealed that, ΔmfsT interrupt the penicillin G (PG) production in M. ruber M7 by blocking the IPN transportation, while PG and IPN produced by the ΔmfsT::mfsT have been recovered the similar levels to those of M. ruber M7. Conclusively, these findings suggest that the M. ruber M7 is, not only a PG producer, but also, indicate that the mfsT gene is supposed to play a key role in IPN intermediate compound transportation during the PG production in M. ruber M7.

Lactobacillus plantarum are amongst the diversified lactic acid bacteria (LAB) species which are being utilized abundantly in the food industry. Numerous L. plantarum strains have been reported to produce several antimicrobial compounds. Diacetyl, hydrogen peroxide, organic acids, as well as bacteriocins can also be exemplified by a variable spectrum of actions. The current study was intended to conduct the screening and characterization of antimicrobial prospective of L. plantarum from traditional Inner Mongolian fermented hard cheese. Foodborne pathogens, Salmonella typhimurium, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus, were examined by using the Oxford cup technique and the mixed culture inhibition assays. The resulting analyses disclosed that L. plantarum KLDS1.0344 indicated broad antimicrobial spectrum against all selected pathogens as compared to other LAB used in this study. Additionally, the decrement of the pathogen population was observed up to 3.47 logs in mixed culture inhibition assays. L. plantarum KLDS 1.0344 acid production was recorded up to 71.8 ± 3.59 °D in mixed culture while antimicrobial particles released in cell free supernatants demonstrated bacteriocin-like characteristics showing substantial pH stability (2.0–6.0), proteolytic enzyme reduced the antibacterial activity (15.2 ± 0.6 mm–20.4 ± 0.8 mm), heat stability (20 min at 120 °C) against selected pathogens. Moreover, the spectrum range of antimicrobial peptides after the partial purification was decreased as compared to the crude bacteriocin-like compound. The SDS-PAGE analysis showed the molecular weight range of partially purified bacteriocin from 12 to 45 kDa. After analyzing the obtained data from the current experimentation showed that the capability of L. plantarum KLDS 1.0344 to oppose the pathogen growth in vitro relies on the occurrence of organic acids along with bacteriocin-like compounds proving L. plantarum KLDS 1.0344 as a potentially appropriate candidate as an alternative bio-control agent against foodborne pathogens.

Polyphenol oxidase (PPO) causes the browning reaction in fruits and vegetables and deteriorates the quality. Thermal treatment for enzyme inactivation may result in defects as opposed to high pressure CO2 (HPCD) processing. In this study, the changes in activity, dissociation, aggregation and conformation of purified PPO from thermal and HPCD treated juice were investigated. HPCD exhibited inactivation of PPO at 55–65 °C whereas thermal processing alone at the same temperature resulted in PPO still showing activity. Under thermal treatment at 25 and 65 °C, the browning degree was higher (0.39 and 0.24) than for HPCD-treated juice (0.23 and 0.12). Fluorescence and circular dichroism spectral results indicated that HPCD induced large decreases in intensities, revealing a rearrangement of the secondary structure and destruction of the native configuration of the PPO molecule. The particle size distribution (PSD) pattern revealed structural modification leading to initial dissociation and subsequent aggregation of PPO after HPCD treatment. Polyacrylamide gel electrophoresis (PAGE) analysis exhibited that molecular size of protein was 40 kDa. In conclusion, the HPCD method was found to be more effective than thermal treatment to inactivate PPO. Structural modifications provided better insights into the phenomena of activation and inactivation of PPO.

Lead (Pb) is a toxic contaminating heavy metal that can cause a variety of hazardous effects to both humans and animals. In the present study, Lactobacillus delbrueckii subsp. bulgaricus KLDS1.0207 (L. bulgaricus KLDS1.0207), which has a remarkable Pb binding capacity and Pb tolerance, was selected for further study. It was observed that the thermodynamic and kinetic model of L. bulgaricus KLDS1.0207 Pb binding respectively fit with the Langmuir–Freundlich model and the pseudo second-order kinetic model. Scanning electron microscopy and energy dispersive spectroscopy analysis disclosed that the cell surfaces were covered with Pb and that carbon and oxygen elements were chiefly involved in Pb binding. Combined with Fourier transform infrared spectroscopy analysis, it was revealed that the carboxyl, phosphoryl, hydroxyl, amino and amide groups were the main functional groups involved in the Pb adsorption. The protective effects of L. bulgaricus KLDS1.0207 against acute Pb toxicity in mice was evaluated by prevention and therapy groups, the results in vivo showed that L. bulgaricus KLDS1.0207 treatment could reduce mortality rates, effectively increase Pb levels in the feces, alleviate tissue Pb enrichment, improve the antioxidant index in the liver and kidney, and relieve renal pathological damage. Our findings show that L. bulgaricus KLDS1.0207 can be used as a potential probiotic against acute Pb toxicity.

Background: Fragile X syndrome, with an approximate incidence rate of 1 in 4000 males to 1 in 8000 females, is the most prevalent genetic cause of heritable intellectual disability and the most common monogenic cause of autism spectrum disorder. The full mutation of the Fragile X Messenger Ribonucleoprotein-1 gene, characterized by an expansion of CGG trinucleotide repeats (>200 CGG repeats), leads to fragile X syndrome. Currently, there are no targeted treatments available for fragile X syndrome. In a recent large multi-site trial, FXLEARN, the effects of the mGluR5 negative allosteric modulator, AFQ056 (mavoglurant), were investigated, but did not show a significant impact of AFQ056 on language development in children with fragile X syndrome aged 3-6 years., Objectives: The current analyses from biospecimens collected in the FXLEARN study aimed to determine whether AFQ056 affects the level of potential biomarkers associated with Akt/mTOR and matrix metalloproteinase 9 signaling in young individuals with fragile X syndrome. Previous research has indicated that these biomarkers play crucial roles in the pathophysiology of fragile X syndrome., Design: A double-blind placebo-controlled parallel-group flexible-dose forced titration design., Methods: Blood samples for biomarkers were collected during the FXLEARN at baseline and subsequent visits (1- and 8-month visits). Biomarker analyses included fragile X messenger ribonucleoprotein-1 genotyping by Southern blot and PCR approaches, fragile X messenger ribonucleoprotein-1 mRNA levels determined by PCR, matrix metalloproteinase 9 levels' detection using a magnetic bead panel, and targets of the Akt/mTOR signaling pathway with their phosphorylation levels detected., Results: This research revealed that administering AFQ056 does not affect the expression levels of the investigated blood biomarkers in young children with fragile X syndrome., Conclusion: Our findings of the lack of association between clinical improvement and biomarkers' levels in the treatment group are in line with the lack of benefit observed in the FXLEARN study. These findings indicate that AFQ056 does not provide benefits as assessed by primary or secondary endpoints., Registration: ClincalTrials.gov NCT02920892., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Dragana Protic has received funding from The Science Fund of the Republic of Serbia and the FRAXA Research Foundation for carrying out studies on Fragile X Syndrome. Flora Tassone has received funding from Zynerba and Azrieli Foundation for carrying out studies on Fragile X Syndrome. Elizabeth Berry-Kravis has received funding from Acadia, Biogen, BioMarin, Erydel, GeneTx/Ultragenyx, Ionis, Jaguar, Kisbee, Neuren, Neurogene, Orphazyme/Kempharm/Zevra, PTC Therapeutics, Roche, Taysha, Tetra/Shionogi, Yamo, Zynerba, and Mallinckrodt Pharmaceuticals, for consulting or on clinical trials in genetic neurological disorders, all funds directed to Rush University Medical Center. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2024.)

Fragile X syndrome (FXS) is the most common heritable form of intellectual disability and is caused by CGG repeat expansions exceeding 200 (full mutation). Such expansions lead to hypermethylation and transcriptional silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene. As a consequence, little or no FMR1 protein (FMRP) is produced; absence of the protein, which normally is responsible for neuronal development and maintenance, causes the syndrome. Previous studies have demonstrated the causal relationship between FMRP levels and cognitive abilities in peripheral blood mononuclear cells (PBMCs) and dermal fibroblast cell lines of patients with FXS. However, it is arguable whether PBMCs or fibroblasts would be the preferred surrogate for measuring molecular markers, particularly FMRP, to represent the cognitive impairment, a core symptom of FXS. To address this concern, CGG repeats, methylation status, FMR1 mRNA, and FMRP levels were measured in both PBMCs and fibroblasts derived from 66 individuals. The findings indicated a strong association between FMR1 mRNA expression levels and CGG repeat numbers in PBMCs of premutation males after correcting for methylation status. Moreover, FMRP expression levels from both PBMCs and fibroblasts of male participants with a hypermethylated full mutation and with mosaicism demonstrated significant association between the intelligence quotient levels and FMRP levels, suggesting that PBMCs may be preferable for FXS clinical studies, because of their greater accessibility., Competing Interests: Disclosure Statement R.J.H. and F.T. received funding from the Azrieli Foundation and Zynerba Pharmaceuticals to perform treatment studies in fragile X syndrome., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Introduction: The chromosome 22q11.2 deletion syndrome (22q11.2DS) is characterized by a well-defined microdeletion and is associated with a wide range of brain-related phenotypes including schizophrenia spectrum disorders (SCZ), autism spectrum disorders (ASD), anxiety disorders and attention deficit disorders (ADHD). The typically deleted region in 22q11.2DS contains multiple genes which haploinsufficiency has the potential of altering the protein and the metabolic profiles., Objectives: Alteration in metabolic processes and downstream protein pathways during the early brain development may help to explain the increased prevalence of the observed neurodevelopmental phenotypes in 22q11.2DS. However, relatively little is known about the correlation of dysregulated protein/metabolite expression and neurobehavioral impairments in individuals who developed them over time., Methods: In this study, we performed untargeted metabolic and proteomic analysis in plasma samples derived from 30 subjects including 16 participants with 22q11.2DS and 14 healthy controls (TD) enrolled in a longitudinal study, aiming to identify a metabolic and protein signature informing about the underlying mechanisms involved in disease development and progression. The metabolic and proteomic profiles were also compared between the participants with 22q11.2DS with and without various comorbidities, such as medical involvement, psychiatric conditions, and autism spectrum disorder (ASD) to detect potential changes among multiple specimens, collected overtime, with the aim to understand the basic underlying mechanisms involved in disease development and progression., Results: We observed a large number of statistically significant differences in metabolites between the two groups. Among them, the levels of taurine and arachidonic acid were significantly lower in 22q11.2DS compared to the TD group. In addition, we identified 16 proteins that showed significant changes in expression levels (adjusted P < 0.05) in 22q11.2DS as compared to TD, including those involved in 70 pathways such as gene expression, the PI3K-Akt signaling pathway and the complement system. Within participants with 22q11.2DS, no significant changes in those with and without medical or psychiatric conditions were observed., Conclusion: To our knowledge, this is the first report on plasma metabolic and proteomic profiling and on the identification of unique biomarkers in 22q11.2DS. These findings may suggest the potential role of the identified metabolites and proteins as biomarkers for the onset of comorbid conditions in 22q11.2DS. Ultimately, the altered protein pathways in 22q11.2DS may provide insights of the biological mechanisms underlying the neurodevelopmental phenotype and may provide missing molecular outcome measures in future clinical trials to assess early-diagnosis treatment and the efficacy of response to targeted treatment., (© 2024. The Author(s).)

The premutation of the fragile X messenger ribonucleoprotein 1 ( FMR1 ) gene is characterized by an expansion of the CGG trinucleotide repeats (55 to 200 CGGs) in the 5' untranslated region and increased levels of FMR1 mRNA. Molecular mechanisms leading to fragile X-premutation-associated conditions (FXPAC) include cotranscriptional R-loop formations, FMR1 mRNA toxicity through both RNA gelation into nuclear foci and sequestration of various CGG-repeat-binding proteins, and the repeat-associated non-AUG (RAN)-initiated translation of potentially toxic proteins. Such molecular mechanisms contribute to subsequent consequences, including mitochondrial dysfunction and neuronal death. Clinically, premutation carriers may exhibit a wide range of symptoms and phenotypes. Any of the problems associated with the premutation can appropriately be called FXPAC. Fragile X-associated tremor/ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), and fragile X-associated neuropsychiatric disorders (FXAND) can fall under FXPAC. Understanding the molecular and clinical aspects of the premutation of the FMR1 gene is crucial for the accurate diagnosis, genetic counseling, and appropriate management of affected individuals and families. This paper summarizes all the known problems associated with the premutation and documents the presentations and discussions that occurred at the International Premutation Conference, which took place in New Zealand in 2023.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.

This study contributes to a greater understanding of the utility of molecular biomarkers to identify clinical phenotypes of fragile X syndrome (FXS). Correlations of baseline clinical trial data (molecular measures- FMR1 mRNA, CYFIP1 mRNA, MMP9 and FMRP protein expression levels, nonverbal IQ, body mass index and weight, language level, NIH Toolbox, adaptive behavior rating, autism, and other mental health correlates) of 59 participants with FXS ages of 6-32 years are reported. FMR1 mRNA expression levels correlated positively with adaptive functioning levels, expressive language, and specific NIH Toolbox measures. The findings of a positive correlation of MMP-9 levels with obesity, CYFIP1 mRNA with mood and autistic symptoms, and FMR1 mRNA expression level with better cognitive, language, and adaptive functions indicate potential biomarkers for specific FXS phenotypes. These may be potential markers for future clinical trials for targeted treatments of FXS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by an expansion of 55-200 CGG repeats (premutation) in the 5'-UTR of the FMR1 gene. Bidirectional transcription at FMR1 locus has been demonstrated and specific alternative splicing of the Antisense FMR1 (ASFMR1) gene has been proposed to have a contributing role in the pathogenesis of FXTAS. The structure of ASFMR1 gene is still uncharacterized and it is currently unknown how many isoforms of the gene are expressed and at what level in premutation carriers (PM) and if they may contribute to the premutation pathology. In this study, we characterized the ASFMR1 gene structure and the transcriptional landscape by using PacBio SMRT sequencing with target enrichment (IDT customized probe panel). We identified 45 ASFMR1 isoforms ranging in sizes from 523 bp to 6 Kb, spanning approximately 59 kb of genomic DNA. Multiplexing and sequencing of six human brain samples from PM samples and normal control (HC) were carried out on the PacBio Sequel platform. We validated the presence of these isoforms by qRT-PCR and Sanger sequencing and characterized the acceptor and donor splicing site consensus sequences. Consistent with previous studies conducted in other tissue types, we found a high expression of ASFMR1 isoform Iso131bp in brain samples of PM as compared to HC, while no differences in expression levels were observed for the newly identified isoforms IsoAS1 and IsoAS2. We investigated the role of the splicing regulatory protein Sam68 which we did not observe in the alternative splicing of the ASFMR1 gene. Our study provides a useful insight into the structure of ASFMR1 gene and transcriptional landscape along with the expression pattern of various newly identified novel isoforms and on their potential role in premutation pathology., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Maternal microbial dysbiosis has been implicated in adverse postnatal health conditions in offspring, such as obesity, cancer, and neurological disorders. We observed that the progeny of mice fed a Westernized diet (WD) with low fiber and extra fat exhibited higher frequencies of stereotypy, hyperactivity, cranial features and lower FMRP protein expression, similar to what is typically observed in Fragile X Syndrome (FXS) in humans. We hypothesized that gut dysbiosis and inflammation during pregnancy influenced the prenatal uterine environment, leading to abnormal phenotypes in offspring. We found that oral in utero supplementation with a beneficial anti-inflammatory probiotic microbe, Lactobacillus reuteri , was sufficient to inhibit FXS-like phenotypes in offspring mice. Cytokine profiles in the pregnant WD females showed that their circulating levels of pro-inflammatory cytokine interleukin (Il)-17 were increased relative to matched gravid mice and to those given supplementary L. reuteri probiotic. To test our hypothesis of prenatal contributions to this neurodevelopmental phenotype, we performed Caesarian (C-section) births using dissimilar foster mothers to eliminate effects of maternal microbiota transferred during vaginal delivery or nursing after birth. We found that foster-reared offspring still displayed a high frequency of these FXS-like features, indicating significant in utero contributions. In contrast, matched foster-reared progeny of L. reuteri -treated mothers did not exhibit the FXS-like typical features, supporting a key role for microbiota during pregnancy. Our findings suggest that diet-induced dysbiosis in the prenatal uterine environment is strongly associated with the incidence of this neurological phenotype in progeny but can be alleviated by addressing gut dysbiosis through probiotic supplementation.

Environmental factors such as diet, gut microbiota, and infections have proven to have a significant role in epigenetic modifications. It is known that epigenetic modifications may cause behavioral and neuronal changes observed in neurodevelopmental disabilities, including fragile X syndrome (FXS) and autism (ASD). Probiotics are live microorganisms that provide health benefits when consumed, and in some cases are shown to decrease the chance of developing neurological disorders. Here, we examined the epigenetic outcomes in offspring mice after feeding of a probiotic organism, Lactobacillus reuteri ( L. reuteri ), to pregnant mother animals. In this study, we tested a cohort of Western diet-fed descendant mice exhibiting a high frequency of behavioral features and lower FMRP protein expression similar to what is observed in FXS in humans (described in a companion manuscript in this same GENES special topic issue). By investigating 17,735 CpG sites spanning the whole mouse genome, we characterized the epigenetic profile in two cohorts of mice descended from mothers treated and non-treated with L. reuteri to determine the effect of prenatal probiotic exposure on the prevention of FXS-like symptoms. We found several genes involved in different neurological pathways being differentially methylated ( p ≤ 0.05) between the cohorts. Among the key functions, synaptogenesis, neurogenesis, synaptic modulation, synaptic transmission, reelin signaling pathway, promotion of specification and maturation of neurons, and long-term potentiation were observed. The results of this study are relevant as they could lead to a better understanding of the pathways involved in these disorders, to novel therapeutics approaches, and to the identification of potential biomarkers for early detection of these conditions.