The cation–chloride co-transporters (CCCs) are the primary systems for maintaining [Cl−]i homeostasis. However, despite extensive electrophysiological data obtained in vitro that are supported by a wide range of molecular biological studies on the expression patterns and properties of CCCs, the presence of ontogenetic changes in [Cl−]i—along with the consequent shift in GABA reversal potential—remain a subject of debate. Recent studies showed that the β3 subunit possesses properties of the P-type ATPase that participates in the ATP-consuming movement of Cl− via the receptor. Moreover, row studies have demonstrated that the β3 subunit is a key player in GABAAR performance and in the appearance of serious neurological disorders. In this review, we discuss the properties and driving forces of CCCs and Cl−, HCO3−ATPase in the maintenance of [Cl−]i homeostasis after changes in upcoming GABAAR function. Moreover, we discuss the contribution of the β3 subunit in the manifestation of epilepsy, autism, and other syndromes.Neuronal intracellular chloride ([Cl The low efficacy of NKCC1 in Cl− uptake has also been observed in sensory neurons. In particular, the use of specific blockers (loop diuretics) of CCCs has indicated that the contribution of NKCC1 to the maintenance of [Cl−]i in these neurons accounts for only 23–36% of total Cl− accumulation i neurons, in which NKCC1 mRNA was expressed, while it had no effect on low-[Cl−]i cells, in which NKCC1 mRNA expression was not detected i and GABAAR-mediated Cl− conductance after changes had occurred was observed over a period of 3–10 s i that is highly hesitant, leading to depolarizing and even excitatory actions under some conditions [− and thus support a low [Cl−]i [−]i occurs after seizures, spinal cord and brain injuries, massive stimulation, and other pathological conditions [−]i accumulation in immature and pathological neurons were explained by the varied efficacy of CCCs [Aergic signaling [−]i, they entirely prevented the secondary rise in [Cl−]i during reoxygenation in hippocampal slices from adult mice [−]i recovery by NKCC1 was observed over 45 min after in vitro ischemia. Kilb et al. demonstrated that bumetanide did not attenuate low-Mg2+-induced epileptiform activity, although it did enhance kainate-induced ictal-like events over 60 min [− accumulation induced by recurrent seizures [AR-mediated current in the presence of bumetanide indicates that a persistent elevation of [Cl−]i in epileptic neurons is only partially mediated by NKCC1 i ,3, whilenditions . Over th of CCCs ,45. Indeignaling . Disruptignaling ,23. Inteult mice , while tr 60 min . Anotherseizures . In addiby NKCC1 .−]i is also considered as one of the major factors in several neurological disorders. In particular, a downregulation of KCC2 associated with epileptiform activity or after an injury has been observed in several in vivo and in vitro studies i in a time scale of seconds via slowly desensitizing or non-desensitizing GABAARs may cause the collapse of the Cl− gradient and contribute to the induction or maintenance of pathological conditions  i homeostasis determines the ambivalent behavior of GABAAR activity. However, its short-term switching and the rapid recovery of GABAAR-mediated Cl− conductance after [Cl−]i changes are difficult to explain simply through the driving force of secondary active cotransporters. The expression of CCCs may be necessary for more delayed and long-term processes for maintaining neuronal [Cl−]i homeostasis, although CCCs are not the decisive factor in short-term and minor changes. In this regard, the existence of an alternative Cl− transport system is necessary to explain the rapid maintenance of Cl− homeostasis.In conclusion, important progress in understanding the molecular mechanisms maintaining chloride homeostasis has been achieved, including a definition of a contribution of specific CCCs in GABAergic signaling. Moreover, these studies reveal the dual nature of GABA action, and i i, the enzyme begins to pump anions into and out of the cell during this time period to reestablish anionic electrochemical gradients for upcoming GABA activity; therefore, this activity may have important physiological significance. Recent research confirms that this ATPase plays an important role in maintaining [Cl−]i levels and shows that enzymatic activity is elevated during seizures [−]i, via the GABAA receptor, contributes to certain disorders  [GABAnd HCO3− ,13,14,150.2/0.3) ,84 can ctivation . Thus, t plateau . To inveseizures  and retuctivity) .ARs possess bifunctional properties—that is, they can operate in two different modes: as a GABAA-gated Cl−-channel or as an anion-transporting P-type ATPase. This indicates that this enzyme not only maintains chloride homeostasis in various neurons, but can also facilitate a change in GABAergic neurotransmission from excitation to inhibition, which is crucial in the CNS  is absolutely necessary for the native assembly of a functional GABAAR . While tAR . This co with α1 ,27,28,29AR depend on the identity of the particular α subunit isoform expressed, with α1-containing receptors having faster kinetics than α2/α3-containing receptors [Drd2) mice [AR β3 subunit gene (GABRB3) is known to play a major role in the development of the CNS, being the major β isoform in multiple regions in the prenatal and neonatal brain [AR function affects neurogenesis and maturation of the neuronal network [Moreover, the decay kinetics of GABAeceptors ,104,105,eceptors ,107. In d2) mice . The GABal brain . A devel network .GABRB3 gene mutations are associated with a broad phenotypic spectrum of epilepsies, and that reduced GABAAR function, causing GABAAergic disinhibition, represents the relevant disease mechanism [GABRB3 in children with epileptic encephalopathies [AR subunit genes  are associated with idiopathic epilepsy, including childhood absence epilepsy, juvenile myoclonic epilepsy, and other syndromes [GABRB3 mutation G32R, which is associated with childhood absence epilepsy, alters the expression of α1β3γ2L GABAAR, as well as channel-gating [GABRB3  impairs GABAAR function [AR coupling junction and pore GABRB3 mutations are linked to early-onset epileptic encephalopathy [AR β subunits (including the β3 subunit) was increased [GABRB3 polypeptides to reduce childhood absence epilepsy [Epilepsy is a group of neurological disorders characterized by recurrent epileptic seizures with a range of etiologies and comorbidities . A numbeechanism ,111,112.opathies . Researcyndromes ,111,114.l-gating . Janve ealopathy ,116. In ncreased . Interesncreased  and reduepilepsy .AR β3 subunit is widely expressed in immature and adult brains in circuits involved in seizure generation, such as the cortex, hippocampus, and thalamic reticular nucleus [AR expression, and seizures that are associated with learning and memory deficits, poor motor skills on a repetitive task, hyperactivity, and a disturbed rest–activity cycle [ARs; pharmacological evidence indicates that other β subunits do not compensate for the absence of β3 [AR β1 and β3 subunits; as a result, Cl− extrusion is impaired, and GABAAR-mediated depolarization appears [The GABA nucleus . Furtherty cycle —all featty cycle ,114,116.ce of β3 . Moreove appears ,123,124.Aergic signaling [ARs in the etiology of ASD was first provided by genetic studies revealing submicroscopic abnormalities in the chromosomal locus 15q11–q13, which contains the GABRB3, GABRA5, and GABRG3 genes encoding the GABAAR β3, α5, and γ3 subunits, respectively [GABRB3 (rs2081648 and rs1426217) genes demonstrates significant associations between ASD and age- and gender-frequency-matched typically developing controls [GABRB3 gene [GABRB3 rs2081648 polymorphisms are associated with symptom-based deficits in social interaction and in sensorimotor and somatosensory coordination, visual response, imitation, activity level, and adaptability [GABRB3 gene was associated with a 3–6 times greater risk of ASD with epilepsy [AR subunits, including the β1 and α4 subunits, has also been coupled with ASD within various ethnic groups [GABRA5 and GABRG3 have a normal phenotype, while GABRB3 knockouts have severe neurological abnormalities, hypersensitivity to tactile stimuli, and defects in social and exploratory behavior [AR subunits (including the ASD-relevant GABRB3) was also altered in the forebrain of young and adult Engrailed-2 knockout mice [ARs are reduced in various brain regions, with GABRB3 significantly altered in the parietal cortex and cerebellum. Overall, β3-containing GABAARs might be important players in the etiology of ASD pathology (Autism spectrum disorders (ASDs) are a group of complex disorders of neurodevelopment characterized by repetitive behaviors and difficulties with social interaction and verbal and nonverbal communication . With ASignaling ,128,129.ectively ,130,131.controls . Locus 1tability . Althougepilepsy , the mutc groups . In addibehavior ,126. Theout mice . In partathology .AR subtypes was altered—α2 was upregulated, while the α5 and β3 subunits were downregulated [AR β2 mRNA in the prefrontal cortex of the AD brain [AR subtypes may have an altered functional profile in AD. This contrasts with the β1 subunit, which was well-preserved.AD is a progressive neurodegenerative disorder that leads to the loss of cognitive functions such as executive function, learning, and memory . AD is aegulated . MoreoveAD brain —along wiAD brain —suggest AR/Cl−, HCO3−ATPase from rat brain is involved in the phenol-induced manifestations of both head-twitching and tremors [ARs via binding with the β3 subunit [PD is the most common neurodegenerative movement disorder. Its clinical manifestations include motor symptoms  and a variety of nonmotor symptoms  ,141. It  tremors . Moreove tremors . In part subunit . In thisARs have been considered as passively permeable, ligand-regulated Cl− channels that—after binding to GABA—mediate either hyperpolarization or depolarization of transmembrane potential. Among all subunits, an exclusive role in GABAAR functioning has been determined only for the β3 subunit. Research data on the properties of GABAARs highlight that the β3 subunit is an independent structure that can singly form an ion pore; importantly, it is also a key polypeptide that facilitates inhibitory neurotransmission. Moreover, recent findings indicate that β3-containing GABAARs are P-type ATPases, allowing us to review them as bifunctional systems. Other β subunits may also possess ATPase activity. These properties of GABAARs necessitate a special look at their role in neurological disorders—for instance, the violation of passive GABAAR-mediated Cl− currents or ATP-dependent Cl− pumping changes chloride homeostasis. In summary, advances in understanding how GABAARs operate have drawn a much more complex picture than what existed previously. New methods (particularly optic genetic methods) and approaches  provide multiple avenues for future research to elucidate the mechanisms of GABAAR function and reveal how its dysfunction leads to disease. We believe that the GABAAR β3 subunit may serve as a primary pharmacotherapeutic target. We hope that this paper will foster interest in clarifying the posttranslational mechanisms of its regulation.Since the 1970s, GABA


Blood-based biomarkers are ideal candidates for dementia prediction. This systematic review and meta-analysis aimed to evaluate longitudinal relationships of blood hormones and hormone-binding proteins in hypothalamic-pituitary (HP) axes with dementia or cognitive decline. PubMed, MEDLINE, EMBASE, PsycINFO, and BIOSIS were systematically searched from 1919 to June 2020. Fifteen types of hormones and four types of hormone-binding proteins were measured in 48 prospective studies. Increased risk of dementia or cognitive decline could be predicted by elevated blood concentrations of free-thyroxine  and sex hormone-binding globulin . Lower thyroid-stimulating hormone (TSH) levels within  and below  the normal range were both risky. Current evidence suggests the alterations of multiple blood molecules in HP axes, especially TSH, free-T4, and SHBG precede the incidence of dementia or cognitive decline. The underpinning etiology remains to be elucidated in the future. Higher proteins  and greaproteins  were detproteins , 12. Theproteins , 13. To Herein, we aimed to evaluate the relationships of hormones and hormone-binding proteins in HP axes with all-cause dementia and cognitive decline based on prospective studies.Out of the 48 eligible articles, 45 were community or population-based, and the other three studies were composed of patients with mild cognitive impairment (MCI) recruited from memory clinics. Total sample size of each axis ranged from 4,626 (HPA axis) to 61,496 (HPT axis). The mean ages of participants in all four axes were above 65 years at baseline, so samples in this review represent predominantly older people. Females were a majority of HPT (62%) axis, a minority of HPG (22%) and HPS (38%) axes and accounted for almost half in HPA (56%) axis. Gender differences in these axes were discussed below. Most studies were performed in western countries, except two in Asia , 35. Abo2I=32.0%). However, the statistical significance was lost when meta-analyzing TSH level across the normal range. In additional analysis, we further proved that it was low but not high TSH level was associated with an increased risk of dementia or cognitive decline, regardless the normal range . Higher free-T4 (the unbound T4 in circulation) also increased the risk of dementia or cognitive decline . Four studies provided gender-specific data. Consistent findings were high free-T4 was detrimental to cognition in male [TSH and free-T4 levels were associated with the risk of dementia or cognitive decline . Higher  in male , 27 whil in male , 24, 27. in male . Egger's2I=24.8%), female  and both genders . Gender differences were observed regarding the effect of gonadal hormones. In female, higher total-E2 was risky , while bio-E2 and free-E2 were not. Higher estrone was also risky in female . In male, none of the androgens was associated with dementia or cognitive decline, though a protective tendency had been detected. Neither estrogens in male nor androgens in female reached statistical significance.IGFBP-2, the most predominant IGFBP in the brain, is the only blood biomarker in HPS axis associated with dementia risk . Result 2I=0.0%).The number of studies and participants regarding blood molecules in HPA axis was limited . Only DH2I <50%), and="" high="" quality="" (NOS=""<7). In this study, we found the increased risk of dementia or cognitive decline could be predicted by a dropped blood concentration of TSH or DHEAS, as well as an elevated blood concentration of free-T4, SHBG, or IGFBP-2. In subgroup analysis, elevated levels estrogen  in female were also associated with the high risk. Among these blood-based biomarkers, evidence of three  was robust on the basis of the large sample size , consistency  and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) groups , 67.We systematically searched PubMed, MEDLINE, EMBASE, PsycINFO and BIOSIS from 1919 to June 29, 2020 without language restriction, using the search terms “hormone”, “thyroid stimulating hormone”, “TSH”, “thyrotropin”, “thyroxine”, “triiodothyronine”, “hypothyroidism”, “hyperthyroidism”, “thyroid”, “follicle stimulating hormone”, “FSH”, “luteinizing hormone”, “LH”, “lactogen”, “pituitary”, “sex hormone”, “androgen”, “testosterone”, “dihydrotestosterone”, “DHT”, “estrogen”, “estradiol”, “estriol”, “estrone”, “progesterone”, “sex hormone binding globulin”, “SHBG”, “growth hormone”, “GH”, “insulin like growth factor”, “IGF”, “corticotropin”, “adrenocorticotropic hormone”, “ACTH”, “dehydroepiandrosterone, “DHEA”, “dehydroepiandrosterone sulfate”, “DHEAS”, “cortisol”, “adrenal”, “dementia”, “Alzheimer”, “cognitive” and “MCI”. The reference lists of eligible articles and relevant reviews were hand-searched for additional citations.Eligible studies had to meet the following criteria simultaneously: (1) had a prospective design ; (2) participants were selected from general populations or memory clinic without dementia; (3) dementia or cognitive decline were recorded by use of well-defined criteria; (4) relative risks (RRs) and 95% confidence intervals (CIs) regarding the association between endogenous blood hormones or hormone-binding proteins in HP axes and dementia or cognitive decline were initially given or could be calculated by information derived from articles. Studies were excluded if they failed to meet any criteria described above. Studies on hormone replacement therapy were not included because this topic had been investigated in recent systematic reviews , 12. If The following information was extracted: first author, publication year, study design, cohort name, source, country, duration of follow-up, follow-up rate, demographic characteristics , exposure , and outcome . The maximally adjusted estimate was applied when several adjusted models were reported in one study. We contacted corresponding authors for further explanation regarding uncertain and missing data. Two investigators independently extracted data. Disagreements were settled by consensus with the help of a third investigator.The Newcastle-Ottawa Scale was used to assess the methodological quality of the included studies. The scale evaluates three domains: selection, comparability, and outcome ascertainment. In this systematic review, we graded the quality as high (7-9 stars), moderate (4-6 stars), and poor (1-3 stars).Risk estimates were expressed as RRs with 95% CIs. A RR above one indicates the high level in blood increases the risk of dementia or cognitive decline, while a RR below one refers to the low level increases the risk. Hazard ratios (HRs) were deemed to be equivalent to RRs. As odds ratios (ORs) often overestimate the true effect, we converted them to RRs using the following formula :0 indicates the incidence of outcome in the reference group or the total sample (when the former is not available). If feasible, RRs and 95% CIs were estimated manually by raw data derived from the text when the original data was not available. We combined the continuous data  with categorical data , and we chose the latter in priority when they were both provided in the same study. For categorical data, if original risk estimates were not presented as the top versus bottom quantile, we graded the bottom quantile as the reference level and recalculated the risk estimate based on a proposed method [Pd method . The univs. normal range, above normal range vs. normal range, the lowest vs. the middle quantile within the normal range, the highest vs. the middle quantile within the normal range. Prespecified subgroup analysis according to gender in HPG axis was conducted in case of the possible sex difference.The free or bioavailable hormones cross the blood-brain-barrier more readily and may be better correlated with cognition . Since s2I statistic. 2I values of 25%, 50%, and 75% represent possibly low, moderate, and high heterogeneity, respectively. Publication bias was evaluated by the Egger's test if there were at least 10 eligible studies. RRs and 95% CIs were log-transformed and pooled using random-effects models. If a study reported data only on Alzheimer’s disease (AD) and vascular dementia (VD) but no data on all-cause dementia, we used the fixed effects model to calculate its RR and 95% CI to estimate the risk of all-cause dementia before the main synthesis. Dementia was preferred if data on dementia and cognitive decline were both available for the same biomarker measured in the same population. Analyses were conducted using Stata version 12.0 and R version 3.5.2.Heterogeneities between studies were assessed by the Supplementary Table 1


Interest in local landraces has unfortunately decreased over, the last decades, in which they have been continuously subjected to a high genetic erosion in favour of new modern varieties. Within the Puglia region , Foggia province was found to be the richest in vegetable landraces. In the present study, six garlic landraces collected from this area have been assessed for their chemical composition  along with their main morpho-biometrical traits. A commercial genotype was also considered as a reference standard. The landraces show a large variability, but in general high morphological standards, high levels of cations and phenols, and low levels of volatile-(S)-compounds in comparison with the commercial genotype and the literature values. ‘Aglio di Peschici’ and ‘Aglio Rosso di Monteleone di Puglia’ are very rich in minerals and phenols (mainly ferulic acid and iso-rhamnetin). This increase in knowledge on the chemical properties of these garlic landraces could represent a tool for encouraging the consumption of a food product. At the same time, the consumption of these landraces would stimulate their cultivation and could highly contribute to protection against the risk of erosion of agro-biodiversity by their in situ/on-farm conservation. Allium sativum L.) is one of the most important bulb vegetables and is mainly used as a spice or flavouring agent for foods. It is used in several types of products such as garlic oil, powder, salt, paste, and flakes.Garlic  compounds and phenolic compounds ,2,3.The organic S-compounds , followed by flavonoids (mainly as quercetin) ,7,8. A lThe Puglia region  in the centre of the Mediterranean basin has a rich agro-biodiversity, with particular reference to landraces . Landraces are variable, identifiable populations, usually known by a local name, lacking ‘formal’ crop improvement, and characterized by a marked adaptation to specific environmental conditions of the area of cultivation .Unfortunately, the regional agro-biodiversity of vegetable crops has been partially lost due to several factors. The modernization and intensification of agriculture have resulted in vast agro-biodiversity loss, since farmers grow modern varieties and hybrids and tend to abandon traditional landraces. https://biodiversitapuglia.it//), aimed at collecting and identifying vegetable resources at risk of genetic erosion, the province of Foggia was found to be the richest in landraces among the five provinces of the region [Alliaceae botanical family the most frequent species found was garlic (Allium sativum L.). To stimulate the cultivation of landraces from the farmers (‘keepers’), their consumption has to be promoted. To do this, the identification and the highlighting of the chemical properties of the landraces compared with the ‘commercial’ varieties may represent an added value of the product that could push the preference of consumers. As a result of a regional Project  were directly collected from local growers which had very small farms in marginal areas, in the province of Foggia, in the North of Puglia region , which lies geographically between 41°54′44′′N and 41°02′27′′N in latitude and 14°55′58′′E and 14°02′27′′E in longitude. All the interested growers had very small farms in marginal areas and all of them adopted low input agronomic practices such as minimum soil tillage, hand weed control and scarce or no use of irrigation, pesticides or fertilizers.Six landraces of garlic (Details of the collected landraces and the collecting sites are presented in Samples of bulbs (3 ± 0.5 kg per each genotype) were well mixed to obtain three independent replicates, each consisting of 15–40 bulbs. Morphological and biometrical measurements on cloves and bulbs were carried out on fresh material, dealing with 7–15 bulbs for each replicate. Chemical analyses of peeled cloves were performed in triplicate for each replicate, after being freeze-dried , powdered, packed in hermetic jars and then stored in the dark at −18 ± 1 °C until the analyses were carried out. The description of the morphological features for each genotype was done following the descriptors 7.1.11 (bulb shape), 7.1.20 (bulb structure type), 7.1.16.1 (bulb outer skin colour), and 7.1.16.2 (skin colour of the clove), provided by the International Plant Genetic Resources Institute (IPGRI) guidelines [Measurements of colour on external bulbs (with dry tunics) and cloves (with thin skin) were also performed by images analysis using an image acquisition station , equipped with four white lamps, camera NIKON D5200, and software Image Pro Plus 7.0 . Colour indices were based on the CIELAB scale 1976: L*, indicating lightness/darkness, ranging from 0 (black) to 100 (white) value in a grey scale; a* reflecting greenish (if negative) to reddish (if positive) tonality; b* indicating bluish (if negative) to yellowish (if positive) tonality. In addition, the derived parameters, hue angle (h°), and chroma (C*), indicating respectively the hue and the vividness/dullness, were evaluated.HPLC-grade methanol, metaphosphoric, formic, methanesulfonic acid, acetonitrile, ethanol were purchased from Sigma-Aldrich . NaOH, sodium carbonate, and sodium bicarbonate were purchased from Merck KGaA . Standards for phenolic  and volatile  compounds were purchased from Sigma-Aldrich . Glucose, fructose, and sucrose standards were purchased from Chem Service ; all the other standards were purchased from Sigma-Aldrich . Ultrapure water (18.2 MΩ cm resistivity at 25 °C) was obtained from a water purification system Milli Q .Ashes were determined by a muffle furnace according to the AOAC method 923.03. −1 HCl in boiling water for 30 min), before injection into the ion chromatography system. For inorganic anions, the lyophilized samples (0.5 g) were extracted with 50 mL of an eluent solution (3.5 mM sodium-carbonate and 1.0 mM sodium-bicarbonate) in a shaking water bath at room temperature for 30 min. The mixture was filtered through a Whatman no. 2 paper. The filtrates were filtered again through a 0.22 µm Millipore filter, before injection into the ion chromatography system.Inorganic ions were analyzed by ion chromatography . Inorganic cations were extracted from lyophilised samples (1 g), previously ashed (in a muffle furnace at 550 °C for 6 h) and acid digested  ; a self-generating DRS-600 suppressor (4 mm), a Dionex IonPack CS12A analytical column , and an eluent solution (20 mM methanesulfonic acid) at a flow rate of 1 mL min−1  .The ion chromatography system was equipped with: an isocratic pump; conductivity detector; and a model AS-DV auto-sampler ; a self-generating ERS-500 suppressor (4 mm), a Dionex Ion-Pac AS23 analytical column , and an eluent solution (3.5 mM sodium-carbonate and 1.0 mM sodium-bicarbonate) at a flow rate of 1 mL minTotal starch was analyzed using the Megazyme Total Starch Assay kit  based on the enzymatic hydrolysis method, following McCleary et al. . LyophilThe starch concentration was determined as liberated glucose. Then, the mixture was centrifuged at 13,000 rpm (10 min); the supernatant was collected and filtered through a membrane filter (0.45 µm).−1 for 15 min. Glucose was identified by a comparison of the retention times with the standard. Peak areas were analyzed using the Dionex Chromeleon software .Samples were analyzed according to Rohrer  using thTotal simple carbohydrates were extracted from 15–30 mg of a lyophilized sample and adding 15 mL of ultrapure water and using shaking water baths  for 45 min at room temperature. Then, the mixture was centrifuged at 13,000 rpm (10 min); the supernatant was collected, filtered through a 0.45 µm membrane filter, and analyzed as previously described. Carbohydrates were identified by a comparison of the retention times with those of sugar standards. Peak areas were analyzed using the Dionex Chromeleon software .The lyophilized sample (0.3 g) was placed in a 50 mL tube and added to 20 mL metaphosphoric acid (0.1%). The sample was mixed in a shaking water bath at room temperature for 15 min, then centrifuged  . The supernatant was collected, filtered, and stored at 4 °C until analyzed according to González-Castro et al.  with somOrganic acids were separated by the ICS 3000 HPLC System  equipped with: An isocratic pump, a 10 µL injection loop, an AS-DV auto-sampler, Hydro-RP 80A column (250 × 4.60 mm) , maintained at 30 °C combined with a UV-visible detector , set to a wavelength of 210 nm and the Chromeleon version 6.50 software.2SO4 at pH 2.6 adjusted with methanesulfonic acid at a flow rate of 1 mL min−1. Individual organic acids were identified by comparing retention times with those of available standards.The eluent consisted of 100 mM Na−1 were placed in a 12 mL screw-cap vial, tightly capped with a PTFE-silicon septum, and conditioned for 10 min at 50 °C. Then, the fiber was introduced into the headspace of the vial for 30 min, removed, and inserted into the gas-chromatography injection port. Desorption of volatiles from the fiber took place in a split mode (1:20 ratio) at 220 °C. The separation of volatile compounds was performed by a Trace 1300 gas chromatograph  equipped with a VF-WAXms capillary column , 20 m length × 0.10 mm ID x 0.1 μm film, and coupled with an ISQ single quadrupole mass spectrometer . The chromatographic conditions were: Oven, 50 °C (0.1 min) to 180 °C at 13 °C min−1, to 220 °C at 18 °C min−1, held for 3 min; detector, source temperature 250 °C; transfer line temperature 250 °C; carrier gas, helium at a constant flow of 0.4 mL min−1. The impact energy was 70 eV. Data were acquired using the full-scan mode in the range of 33 to 280 m/z at an acquisition rate of 7.2 Hz.Volatile compounds were extracted by the HS-SPME technique using a DVB-CAR-PDMS fiber  according to Gambacorta et al. , with soVolatiles were identified by comparing the experimental spectra with those obtained by the available pure standard compounds and with those reported in the NIST Library  and quan2; sheath gas flow 30 arbitrary units; auxiliary gas flow 15 arbitrary units; capillary voltage—2.8 kV, S-Lens RF Level 60%. Data were acquired in a negative ionization mode.The lyophilized sample (0.05 g) was placed in a 2 mL Eppendorf tube and added to 1 mL of 80% methanol in water. The sample was mixed for 1 min, sonicated for 5 min, and then centrifuged (4000 g 4 °C for 20 min). The clear supernatant was diluted 1:1 with an acetonitrile:water  solvent mixture containing 0.1% formic, and filtered using re-generated cellulose filters of 0.22 μm pore diameter. The analysis was performed according to Pasqualone et al.  with somPhenolic compounds were identified by comparing elution times, molecular ions, MS/MS fragmentation patterns of the experimental spectra with those obtained by the available pure standard compounds or by tentative methods using reported data from the literature. Calibration curves were created to obtain quantification results and were based on the UV signal of each available standard. When no commercial standard was available, a similar compound from the same phenolic group was used as a standard.p = 0.05) was used to establish differences between means. A one-way analysis of variance was performed using the Statistical Analysis Software . The least significant difference (LSD) test (http://folk.uio.no/ohammer/past) [For a visual analysis of data, the Principal Component Analysis (PCA) was performed using the PAST3 Software (er/past)  on mean Refer to According to IPGRI descriptors, the Commercial Genotype (CG) used in this study was grouped as a ‘flat globe’ type with a ‘regular multi-cloved radial’ bulb structure. Among the landraces, the bulb shape ranged from ‘broad oval’ for ‘Panni’, ‘PanniD’, and ‘Monteleone’, to ‘flat globe’ for ‘Anzano’ and ‘Cortigli’ and to ‘globe’ for ‘Peschici’. All the landraces studied had an ‘irregular’ bulb structure, except for ‘Peschici’, which was in the ‘regular two-fan group’ .Concerning the colour of bulbs (with dry tunics) and cloves (with thin skin) , all lanAglio Durevole di Panni’ meaning a long storage life. PCA also discriminates ‘Peschici’ from the other genotypes for the highest number of cloves per bulb (Number-c) (highly and positively correlated to PC1) and by the lowest weight of cloves (W-c) (highly and positively correlated to PC2).A high variability was observed in biometrical traits of both bulbs and cloves . The Priagronomic) characters, even between genotypes grown in the same areas for a long time. This is probably mostly a consequence of the accumulation of natural mutations and their evolving over time due to the selection pressures and cultural data in the minds of local farmers [This high variability in morpho-biometrical traits in the examined landraces is, of course, the result of the long process of (un)conscious selection made by farmers and of their continued evolution in a certain eco-geographical area . Althoug farmers ,20. The variability of the morpho-biometrical traits of garlic genotypes has been widely reported ,21. The The concentration of ashes, cations, and anions is reported in −1 dry weight—dw) and cations (15.6 g kg−1 dw), particularly K. ‘Cortigli’ along with ‘PanniD’ had the highest Mg concentration, whereas ‘Anzano’ had the highest Ca concentration. Regarding anions, the total concentration in the landraces  was in line with that of the CG; ‘Peschici’ showed the highest values of all the individual anions, followed by ‘Cortigli’ . Among the examined landraces, ‘PanniD’ was the poorest in all anions and cations, except for Mg. The content of ash and cations ranged from the highest values in ‘Peschici’ and ‘Monteleone’ to the lowest in ‘PanniD’. On average, landraces were richer than the CG in ash  was slightly higher than the values reported in Greek garlic landraces [−1 fw, on average) can be considered in line with those reported in official food composition databases, such as that of the European Food Safety Authority (EFSA) (579 mg 100 g−1 fw) and of the Italian National Centre of Agriculture (INRAN-CREA) (600 mg 100 g−1 fw), and higher than those reported by the United States Standard References (USDA) (401 mg 100 g−1 fw). The levels of K in our landraces were similar to those found in Greek [The amount of ash concentration in our landraces . ‘Panni’, as well as the CG, showed the highest level of total simple carbohydrates, mainly due to the contribution of sucrose. The CG had the highest value of fructose, followed by ‘Monteleone’ and ‘Anzano’. Since extensive studies on several vegetables have correlated total simple sugars with perceived sweetness [Genotypes differed distinctively in the amount of total carbohydrates and individual soluble sugars, except for glucose  was lower than that of the CG (−1 fw) or the USDA (1.0 g 100 g−1 fw) databases.On average, the total carbohydrate content in our landraces , followed by fructose (10%) and glucose (5%) . Sucrose−1 dw; 0.054 g 100 g−1 fw) was lower than that of the CG and other genotypes [−1 fw), while fructans have been detected in a larger proportion [Allium spp. bulb tissue could be attributed mainly to fructans.Concerning the starch concentration , ‘Anzanoenotypes ,29. AlthThe organic acid concentrations of the studied garlic genotypes are presented in −1 dw; 1.0 g 100 g−1 fw) were lower than that in the CG (−1 fw) and from Latvia (3.87 g 100 g−1 dw) [On average, the total organic acid contents in our landraces  .In the examined landraces, the organic acid profile was represented by 43% citric, 21% malic, 18% oxalic, and 18% pyruvic acid . The org−1 fw) and in the USDA (31.2 mg 100 g−1 fw) food composition databases. In the collected landraces, and particularly in ‘Monteleone’, ‘Peschici’, and ‘Anzano’, ascorbic acid  was distinctively higher than in the CG . HoweverThe volatile compound concentration in the studied garlic genotypes is reported in The precursors of the volatile organic S-compounds in garlic are γ-glutamyl cysteine and cysteine sulfoxides, including deoxyallin and mainly alliin  , an odouIn the current study, the main fraction of volatile substances was the S-compounds , with the non-sulphur compounds  representing only a minor fraction . Among the examined genotypes, a total of 28 compounds were identified in the volatile fraction, 16 of them were S-compounds, while the other compounds were hydrocarbons, alcohols, and ethers .−1 dw, on average), mainly due to the amount of S-compounds . Among the non-sulphur volatile compounds, a lower concentration of total aldehydes  and ‘other’ compounds  was found in the examined landraces. In particular, the landraces were completely devoid of two out of four of the determined aldehydes—‘2-butenal-2-methyl’ and ‘4-heptenal’. The total volatile concentration in CG bulbs was distinctively higher than those of landraces  (among the S-compounds), and 4-heptenal-enantic aldehyde , were detected in very low amounts (<0.01 unit).Except for the 1-propanethiol S-compound, the genotype distinctively affected the concentration and the profile of volatile compounds.−1 dw—total volatile compounds; 12.0 vs. 2.0 mg kg−1 dw—S-compounds). Apart from the genotype, other factors such as ‘environment’ may affect volatile compound concentrations, as widely proved in the specific literature on garlic [A large variation in the amount of (S)-volatile compounds was observed between the GC and the collected landraces , followed by 3-vinyl-1,2-dithyocyclohex-5-en’ , di-allyl-disulphide (DADS) , 3-vinyl-1,2-dithiin , and methyl-allyl-tri-sulphide . The quantitative analysis in the CG showed that five compounds represented 85% of the total S-compounds: 3-vinyl-1,2-dithyocyclohex-5-ene , 3-vinyl-1,2-dithiin (18%), DATS (10%), and DADS (8%) and methyl-2-propenyl-di-sulphide (8%).In the current study, in the examined landraces the five most abundant S-compounds (representing 93% of the S-compounds) were ‘di-allyl-trisulphide’ (DATS)  indicating an intense pungent odour ,43. Acco−1 dw, on average) was the most abundant, followed by the caffeic acid derivates—caffeic acid-O-hexoside-1  and caffeic acid-O-hexoside-2 . The flavonoid component (six compounds), represented 16% of the total phenols, and among them, iso-rhamnetin  and rutoside-1  were the most abundant. Less than 0.3% of total phenols were unknown compounds. A 3-hydroxy-methyl phenol was found only in the CG. Among the flavonoids, quercetol (quercetin), patuletin, hyperoside (hyperin), iso-quercitrin (quercetin 3-β-D-glucoside), and coumaroyl-quinic acid among the phenolic acids were detected at a very low rate (<0.5 unit). Phenolic compounds are reported in Except for caftaric and caffeic acid-O-hexoside-1 acids, the genotype distinctively affected the concentrations and the profile of phenols. −1 dw), due to the high contribution of both components. The CG showed a very small amount of flavonoids (only quercetol). On average, the phenolic concentration in our landraces was much higher than in the CG  [−1 dw) [−1 dw) and in flavonoids (29.9 mg kg−1 dw) [The levels of phenolic compounds in our landraces were also substantially greater than that reported for Polish garlic (11.5 mg kg−1 dw)  and part [−1 dw) , as wellkg−1 dw) .The antioxidant, antibacterial, anti-inflammatory, anti-proliferative, and chemopreventive properties of phenolics and flavonoids in vegetables and Allium spp. ,48 are w−1 dw in Almeria [−1 dw in Andalusia [−1 dw [Among the examined landraces, ‘Peschici’ and ‘Monteleone’, followed by ‘Cortigli’ and ‘Anzano’ showed the highest total phenol concentrations. The most abundant compounds were ferulic acid along with caffeic acid-O-hexoside-2 (phenolic acids) and iso-rhamnetin and rutinoside-1 (flavonoid compounds). In this study, we found significantly higher amounts of ferulic acid both in comparison with the tested GC and also compared with those found in several genotypes in Spain −3.5 mg kg Almeria ; 0.9–7.3ndalusia ; and in a [−1 dw , has beea [−1 dw , stem boa [−1 dw . TherefoHydroxycinnamic acids as well as ferulic and caffeic acids have been reported, similar to many phenols, as antioxidants since they are reactive toward free radicals as a reactive oxygen species . CaffeicAllium neapolitanum and evaluated for its anti-aggregation human platelet activity by Carotenuto et al. [Hippophae rhamnoides L.), has been shown to play a protective role against acute fulminant hepatitis in mice [Concerning iso-rhamnetin, it was identified as a new flavonoid glycoside in o et al. . Recentl in mice .Allium spp. , has been shown to have anti-proliferative potential in various cancer cell lines [‘Peschici’ and ‘Monteleone’ appreciably highlighted a high amount of rutoside-1, known as rutin (a flavonol glycoside between quercetin and disaccharide rutinose), which has been found to have important pharmacological effects, such as in the prevention of ulcerative colitis . Both ‘Pll lines .Allium spp. from Romania  Holub; A. schoenoprasum L. subsp. Schoenoprasum) [Urtica urens L., patuletin has been shown to possess an antioxidant activity and free radical scavenging effects in rats treated with aflatoxin B1, a risk factor for hepatocellular carcinoma [‘Anzano’ along with ‘Monteleone’ were the only two genotypes containing patuletin, a compound also detected on oprasum) . Isolatearcinoma . Furtherarcinoma .Among the examined landraces, those from Panni Municipality were the poorest in total phenolic compounds but contained a good level of caffeic acid-O-hexoside-1 acid (phenolic acid) and iso-rhamnetin and rutoside-1 (flavonoids). Among the investigated genotypes, ‘PanniD’ showed the highest value of hyperoside (hyperin) and it was the only landrace which accumulated a considerable amount of quercetol (quercetin). Quercetol (quercetin) has important functional benefits, including an anti-inflammatory activity, an anti-histamine effect, allergy medication, as well as anticancer and antivirus activities. It has also been claimed to regulate blood pressure in hypertensive subjects . The hypThe results of the PCA using the chemical parameters showed that the first two PCs explained approximately 70% of the total variability, attributing 42% to PC1 and 26% to PC2 . The group of ‘Anzano’, ‘Monteleone’, ‘Cortigli’, and ‘Peschici’ landraces separated from the ‘PanniD’, ‘Panni’ landraces (low side of PC2) and also from the CG (left side of PC1). The former group clustered for the concentration of phenolic compounds  (highly and positively correlated to PC1) and for the high concentration of anions and cations (highly and positively correlated to PC2). ‘Peschici’ and ‘Monteleone’ set in an extreme position of this quadrant, mainly characterized by the highest mineral (K) concentration and phenols, as also underlined by Anova . ‘AnzanoConversely, the CG tended to cluster separately on the left side of PC1 for the general negative correlation with phenolic compounds and for the positive correlation with the volatile S-compounds , with a phenolic compound as Quercetol (Quertecin) , exclusively present in ‘PanniD’ .productive) standard features for the market, in line with those of the examined commercial genotype used as a reference standard.The observed diversity in the morpho-biometrical and chemical traits among the examined landraces can be explained by considering the long process of adaptation to the specific ‘environment’ (pedo-climatic properties and less intensive agronomic practices), along with the constant selection pressure performed by farmers over a long period. These factors ensured a ‘non-homologation’ of the final product and specificity of the morpho-biometrical and chemical characteristics of a single genotype. Findings highlighted that the landraces collected from the province of Foggia maintain high biometrical (+) and phenols . The examined landraces were able to accumulate nutrients and phytochemicals, thus showing interesting features for the human diet. They exhibited high total cations (mainly KBy increasing the knowledge of the properties of these local garlic landraces, this work could represent a key tool for the promotion of their consumption. At the same time, the consumption could encourage their cultivation, thus contributing to the protection of this agro-biodiversity at a high risk of genetic erosion by promoting their in situ/on-farm conservation.


Cbs gene in mice causes severe hyperhomocysteinemia and reduces life span. Here, we examined a possible involvement of senescence, mitochondrial DNA, and telomeres in the reduced life span of Cbs−/− mice. We found that senescence-related p21, Pai-1, Mcp1, and Il-6 mRNAs were significantly upregulated (2–10-fold) in liver, while p21 was upregulated in the brain of Cbs−/− mice (n = 20) compared with control Cbs+/− siblings (n = 20) in a sex- and age-dependent manner. Telomere length in blood (n = 80), liver (n = 40), and brain (n = 40) was not affected by the Cbs−/− genotype, but varied with sex and/or age. Levels of mitochondrial DNA tended to be reduced in livers, but not brains and blood, of Cbs−/− females (n = 20–40). The Cbs−/− genotype significantly reduced Tert mRNA expression in brain, but not liver, in a sex- and age-dependent manner. Multiple regression analysis showed that the senescence-related liver (but not brain) mRNAs and liver (but not brain or blood) mitochondrial DNA were associated with the Cbs genotype. In contrast, telomere length in blood, brain, and liver was not associated with the Cbs genotype or hyperhomocysteinemia, but was associated with sex (in brain and liver) and age (in brain and blood). Taken together, these findings suggest that the changes in senescence marker expression and mtDNA levels, but not telomere shortening, could account for the reduced life span of Cbs−/− mice.Cystathionine β-synthase (CBS) is a housekeeping enzyme that catalyzes the first step of the homocysteine to cysteine transsulfuration pathway. Homozygous deletion of the  Cellular senescence irreversibly arrests proliferation and is an important contributor to aging and age-related disease . SenesceTelomeres are tandem DNA repeats of TTAGGG structures at the end of chromosomes important for genomic stability in all vertebrates. The telomere sequence is more sensitive to damage than non-telomeric DNA, and its length progressively shortens with age during somatic cell division. Short telomeres are a feature associated with cellular senescence and death. Telomeres are synthesized by the telomerase (TERT) enzyme, a reverse transcriptase active during early human development, but becoming silenced in somatic cells at 12–18 weeks of gestation . HoweverMitochondrial DNA (mtDNA) is an essential multicopy organellar genome organized into protein-DNA structures called nucleoids. The mtDNA lacks histones and is more sensitive to damage than nuclear DNA . Like teCBS gene, is characterized by severely elevated levels of homocysteine (Hcy) and its metabolites [CBS−/− patients are mentally retarded and experience thromboembolic events in brain, heart, and peripheral veins and arteries, which contribute to the reduced life span in these individuals [Cystathionine β-synthase (CBS) deficiency, a rare metabolic disease caused by mutations in the abolites ,21,22,23abolites ,25. Thesividuals ,25.Cbs gene leads to a neonatal lethality phenotype due to liver dysfunction with only a few percent of Cbs−/− pups surviving to adulthood [Cbs−/− mice harboring wild-type or mutant versions of the human CBS gene under the control of the zinc-inducible metallothionein promoter prevents the neonatal lethality. One of those variants, the Tg-I278T Cbs−/− mouse, has a median survival time 25% shorter compared to sibling controls (613 vs. 821 days) [Homozygous deletion of the dulthood . The creTert and senescence/aging marker  mRNA expression in the tissues of severely hyperhomocysteinemic Tg-I278T Cbs−/− mice and their sibling controls.Although Cbs deficiency reduces life span, it is not known whether telomere shortening, senescence, and/or mtDNA copy number are involved. For this reason, the present work has been undertaken to study telomere length (TL), mtDNA levels, and Tg-I278T Cbs−/− mice have severe hyperhomocysteinemia (HHcy) , and endoplasmic reticulum stress in liver and kidney [siblings ), a 25% siblings  and a resiblings . Other cCbs−/− mice could be caused by premature senescence. To examine this hypothesis, we quantified the expression of senescence-related mRNAs  and an aging-related Kl mRNA relative to β-actin and Gapdh mRNAs in mouse brains and livers. We found that Pai-1, p21, Mcp1, and Il-6 mRNAs were significantly upregulated in livers of Cbs−/− mice compared to Cbs+/− siblings (p16 and Il-1β mRNAs were not affected by the Cbs−/− genotype (Cbs−/− mice and their Cbs+/− siblings (not shown).We hypothesized that the reduced life span of the siblings A–C,E, whgenotype D,F. The Cbs−/− genotype on the expression of senescence-related mRNAs were sex- and age-dependent. In young Cbs−/− female mice, liver p21, Pai-1, and Mcp-1 mRNAs were significantly upregulated  compared to young Cbs+/− females. Liver p21 and Pai-1 mRNAs were also upregulated  in old Cbs−/− females  compared to young Cbs+/− males. Liver Mcp-1 mRNA was also upregulated  in old Cbs−/− males  and Il-6 mRNAs  (Sex significantly affected the expression of liver = 0.010) .Cbs−/− mice, age significantly affected the expression of liver Pai-1  and Mcp-1 mRNAs . In Cbs+/− mice, age affected the expression of liver Mcp-1, Il-6  and p21 mRNAs   .Cbs genotype had no effect on Il-1β and p16 mRNAs in the whole mouse cohort , but not in males. Il-1β mRNA was not affected by the Cbs genotype in any of the age and sex subgroups. Il-1β mRNA was significantly upregulated in old mice, similarly in Cbs−/− and Cbs+/− females and males .Although the e cohort D,F, subgp21, Mcp-1, Pai-1, and Il-6 mRNAs appeared to be unaffected by the Cbs−/− genotype in the whole mouse cohort, as was Kl mRNA (not shown). However, examination of sex and age subgroups revealed that, in Cbs−/− female mice, brain p21 and Mcp-1 mRNAs were significantly upregulated relative to Cbs+/− females . In Cbs+/− mice, sex affected the expression of brain p21 in old mice  and Mcp-1 mRNAs , Kl , and p21  mRNAs (Age significantly affected the expression of brain 8) mRNAs .Il-1β and p16 mRNAs were not affected by the Cbs genotype in any of the age and sex subgroups. Age and sex did not affect brain Il-1β mRNA, while age significantly affected brain p16 mRNA .Brain Cbs−/− mice (n = 40) and their Cbs+/− siblings (n = 40) and found that blood TL was not affected by the Cbs genotype  and males . Blood TL was also similar between old Cbs−/− and Cbs+/− mice of both sexes   .Cbs−/− compared to Cbs+/− mice  , but not in males, and were significantly higher in females compared with males   B, as exp= 0.044) . Notably= 0.044) A and in = 0.044) , blood TCbs−/− mice, blood TL was significantly reduced in older compared to younger animals    . Similar< 0.000) . In contblood TL .Cbs+/− and Cbs−/− mice. Specifically, blood TL was significantly higher in old Cbs+/− and Cbs−/− females compared with old males  (Cbs+/− and Cbs−/− mice.The effect of sex on blood TL was similar between < 0.000) . HoweverCbs genotype affects TL in a tissue-specific manner, we isolated DNA from brains and livers of Cbs−/− mice (n = 19) and their Cbs+/− siblings (n = 24) and quantified brain and liver TL. To examine whether TL is affected by telomerase expression, we isolated RNA and examined Tert mRNA levels in these tissues.To examine whether Cbs genotype . In contrast, in young and old Cbs+/− mice, brain TL was not affected by sex and was similar between males and females   .Tert mRNA expression was also affected by sex. Specifically, in young Cbs−/− mice, brain Tert mRNA was significantly elevated in females compared to males  .Brain = 0.017) . In old Tert mRNA expression was also affected by age in a sex-dependent manner. Specifically, brain Tert mRNA levels were significantly reduced in old compared to young male , but not female, Cbs+/− mice; however, this effect of age was absent in Cbs−/− mice  and Cbs+/− mice   . In contnd males .Cbs−/− and Cbs+/− mice  . In old Cbs+/− mice, liver Tert mRNA was higher in females compared to males   . In old = 0.012) .In the whole mouse cohort, blood, liver, and brain mtDNA levels were not affected by the Cbs genotype . HoweverCbs−/− and Cbs+/− mice of each sex are shown in Cbs−/− and Cbs+/− mice and for both females and males.Relationships between blood mtDNA and age for Cbs genotype, but was affected by sex and age: higher in young compared with old female Cbs−/−  and Cbs+/−  mice. mtDNA was also higher in young vs. old male Cbs−/−  and Cbs+/−  mice  mice .Cbs+/− mice: higher in young males compared with young females . This sex effect was abrogated by the Cbs−/− genotype   .Cbs−/− mice compared to Cbs+/− animals , while brain mtDNA was not affected by the Cbs genotype  and Cbs+/− males . Increased mtDNA levels were also found in livers of old compared to young Cbs−/− and Cbs+/− mice of both sexes.We found that brain and liver mtDNAs were affected by sex  and age . HoweverCbs−/− and Cbs+/− mice showed that blood TL was significantly associated with age, but not with the Cbs genotype or tHcy , sex (p = 0.021), and age (p = 0.025) (p = 0.007) and age (p = 0.005). Liver mtDNA was associated with age.Pearson correlation analysis of the whole cohort including = 0.025) . Brain mCbs genotype (p = 0.014) and age (p = 0.005) in males. Brain and liver TL were associated with age in males and females.Pearson correlation analysis stratified by sex showed that blood mtDNA was significantly associated with the Cbs genotype or plasma tHcy, liver mtDNA was significantly associated with the Cbs genotype  and age  in the whole mouse cohort including both sexes. The association with the Cbs genotype was somewhat stronger in the female subgroup . In contrast, in the male subgroup, age (p = 0.003), but not the Cbs genotype, was the only significant determinant of liver mtDNA. The Cbs genotype and age explained 44% of the liver mtDNA variability in the whole cohort and 63 % in the female subgroup  and 24–29% (blood) of the mtDNA variability (p = 0.023) and male  subgroups.In contrast, brain and blood mtDNAs were not associated with the iability . Brain mCbs genotype or plasma tHcy showed that most of the blood mtDNA variability occurred in males (R2 = 0.35–0.48), not females (R2 = 0.01–0.1). The blood mtDNA was strongly associated with age in males  and tended to be associated with the Cbs genotype (p = 0.058), but not plasma tHcy (p = 0.419). Age and the Cbs genotype explained 48% of the variability in mtDNA in males , while liver mtDNA was the strongest determinant of liver Il-6 . In addition, Tert mRNA (p = 0.002–0.006) was a positive determinant of liver Pai-1, Mcp1, and Il-6 mRNAs, while liver mtDNA (p = 0.002) was a positive determinant of liver Pai-1 and Il-6 mRNAs in females  and Mcp1 (p = 0.013) mRNAs. In addition, liver Tert mRNA (p = 0.020) and age (p = 0.002) were positive determinants of liver Pai-1 and Mcp1 mRNAs, respectively; liver Tert mRNA (p = 0.016) was a negative determinant, while liver mtDNA (p = 0.001) and age (p = 0.015) were positive determinants of liver Il-6 mRNA  of Pai-1 mRNA, sex (p = 0.044), and TL (p = 0.028) of p21 mRNA  and age (p = 0.008) and Tert mRNA (p = 0.019) of Il-6 and Kl mRNAs, respectively  were significantly upregulated in livers of Cbs−/− mice compared to control Cbs+/− siblings . However, although SA-β-gal is prominent in senescent sells, it is neither required, nor a determinant of the senescence phenotype [Cbs−/− and Cbs−/− mice was 20 to 40 per each Cbs genotype group, the number of animals in the sex- and age-stratified subgroups was accordingly much lower. However, despite the relatively low number of animals in these subgroups, we found that age and sex, which are known to affect TL [The limitations of this study should be noted. First, we did not study the expression of senescence-associated genes at the protein level. Measuring senescence-related protein levels in the mouse  can be challenging mostly due to the availability of antibodies . Howeverhenotype ,37. Thirffect TL ,15, wereffect TL  and ratsffect TL , was assCbs−/− mice had significantly upregulated expression of some senescence-related mRNAs  in liver, but not in brain, and reduced mtDNA copy number in liver, but not in brain and blood. We also showed that TL in any of those tissues was not affected by the Cbs−/− genotype. These findings suggested that accelerated senescence, mostly affecting liver, and impaired mitochondrial function, but not telomere shortening, could contribute to the reduced life span of Cbs−/− mice.In conclusion, we showed that Tg-I278T Cbs−/− mice on a C57BL/6J genetic background [Tg-I278T Cbs+/- counterparts to generate sufficient numbers of Tg-I278T Cbs−/− and Tg-I278T Cbs+/− animals required for the experiments. The mice were bred and housed at the New Jersey Medical School Animal Facility [CBS-I278T variant was under the control of the zinc-inducible metallothionein promoter, which prevents the neonatal lethality of the mouse Cbs−/− genotype by supplementation of the drinking water with 25 mM ZnCl2 [Cbs genotype was established by PCR using the following primers: forward 5’-GGTCTGGAATTCACTATGTAGC-3’, Cbs- reverse 5’- GAGGTCGACGGTATCGATA-3’ (affording a 176 bp product), Cbs+ reverse 5’-CGGATGACCTGCATTCATCT-3’ (affording a 300 bp product). Mice (n = 40 to 80) of both sexes, 63 to 408 days old, were used in experiments. Animal procedures were approved by the Institutional Animal Care and Use Committee at the New Jersey Medical School.Transgenic Facility ,38. In tv/v) 0.5 M EDTA. After centrifugation , separated plasma and cells were frozen at −80 °C. Mice were euthanized using CO2 and the organs collected and frozen on dry ice. Brains and livers were powdered with dry ice using a mortar and pestle and stored at −80 °C.Blood was collected from cheek veins into Eppendorf tubes containing 1% , post-column derivatized with escribed ,39. An ATotal DNA, extracted from brain, liver, or whole mouse blood using the phenol method, was treated with RNase A  and diluted with ultrapure water. The integrity and quality of the DNA were tested by agarose gel electrophoresis and spectroscopy .4GGTTAGTGT-3’; reverse 5’-TGTTAGG-(TATCCC)5TAACA-3’). The amplification of the single copy gene albumin  was used as a control for the input DNA [Mouse genomic DNA was extracted from the whole blood using the phenol extraction method and stored at −80 °C. The telomere length assays were performed using the quantitative polymerase chain reaction (PCR) method to measure telomere length relative to standard reference DNA (T/S ratio), as described in detail elsewhere ,41. The nput DNA ,41.® Green Supermix , 900 nM of telomere- or albumin-specific primers, and 160 ng DNA as a template. The optimized thermal cycling profile for each reaction mixture was as follows: Step 1: 94 °C for 15 min.; Step 2: 94 °C for 15 s, 49 °C for 15 s, two cycles; Step 3: 94 °C for 15 s, 62 °C for 10 s, 74 °C for 15 s with signal acquisition, 84 °C for 10 s, 88 °C 15 s with signal acquisition, 35 cycles; Step 4: 65–95 °C with signal acquisition . The 74 °C reads provided the cycle threshold (Ct) values for the amplification of the telomere template, and the 88 °C reads provided the Ct values for the amplification of the albumin. All qPCRs were carried out on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Polska Sp. Z o.o.).The master mix for each PCR amplification tube was prepared with iTaq™ Universal SYBRCt, the Ct for the single-copy albumin gene was subtracted from the telomere Ct . To calculate ΔΔCt, the ΔCt for the DNA from control Cbs+/− individuals was averaged and subtracted from the ΔCt for the DNA from each Cbs−/− mouse and Cbs+/− control animal (ΔΔCt = ΔCtCbs−/− − ΔCtCbs+/−). The T/S values were calculated according to the T/S = 2−(Ct)ΔΔ equation [The relative telomere length (TL) was calculated from the Ct values as the ratio of the telomere signal to the single copy albumin gene signal (T/S ratio) ,41. To cequation . Each saDNA was diluted with ultrapure water and sonicated in an ultrasonic bath  prior to qPCR analysis . The mtDAll reactions were performed in 10 µL volumes in duplicate using 20 ng DNA, 3 µM primers, and 5 µL iTaq Universal SYBR Green Supermix (Bio-Rad Polska Sp.z o.o.) using a CFX96 Touch Real Time PCR Detection System (Bio-Rad). Cycling parameters: 95 °C (10 min), 40 cycles at 95 °C (15 s), 60 °C (10 s), and 72 °C (15 s). The homogeneity of qPCR products was confirmed by melting curve analysis (65 °C and a progressive increase up to 95 °C at 0.5 °C/min). Analysis of the data was performed with the CFX Manager™ Software and Microsoft Excel. Calculations were similar as those for telomere length above .Total RNA was extracted using a column-based Total RNA Purification Kit . Three-zone reagent was added to the frozen powdered brain or liver tissue. After the extraction step, the supernatant was mixed with 70% ethanol, loaded onto a total RNA miniprep column, washed with 70% ethanol, and eluted with ultrapure water. The RNA was DNase treated using the DNase I . The integrity and quality of the RNA were confirmed by agarose gel electrophoresis and spectroscopy .23  .cDNA synthesis was carried out in reaction mixtures (20 µL) containing 1 μg of total RNA, RivertAid H Reverse Transcriptase , and oligo(dT)Tert) or 40 ng (for senescence markers) cDNA, 0.3 µM primers , and the anti-aging protein Klotho (Kl) were normalized to β-actin and Gapdh mRNAs. Data analysis was performed with the CFX Manager™ Software, Microsoft Excel, and Statistica. The 2Ct) containing 20 ng .Data are expressed as the means ± SEM  and the


Background: The purpose of this study was to evaluate the implementation and effectiveness of an ecological, multi-component adolescent obesity prevention intervention called School Wellness Integration Targeting Child Health–Middle School (SWITCH–MS). Methods: Following the effectiveness-implementation hybrid type 3 quasi-experimental design, seven middle schools (377 students) in Iowa, United States, were stratified into “experienced”  or “inexperienced”  groups to receive the 12-week SWITCH–MS intervention. To evaluate implementation, school informants (n = 10) responded to a survey and students completed behavioral tracking in the classroom on a website. For effectiveness evaluation, students in 6th, 7th, and 8th grades completed a validated questionnaire before and after intervention, to measure behaviors of physical activity , screen-based activity (“View”), and fruits and vegetable consumption (“Chew”). Results: The two groups of schools showed similar levels of implementation for best practices, awareness, and engagement. Behavioral tracking rate favored the experienced schools early on (47.5% vs. 11.7%), but differences leveled off in weeks 3–12 (sustained at 30.1–44.3%). Linear mixed models demonstrated significant time effects for “Do”  and “View” behaviors (p = 0.02), after controlling for student- and school-level covariates. Conclusions: This study demonstrates that prior experience with SWITCH–MS may not be a prominent factor for implementation and effectiveness, although greater experience is associated with favorable behavioral tracking when the intervention is first launched. Childhood obesity remains a major global public health concern with over 340 million children and adolescents being classified overweight or obese in 2016 ,6. TheseThe School Wellness Integration Targeting Child Health (SWITCH) is an evidence-based childhood obesity prevention intervention (www.iowaswitch.org), centered on building school capacity to implement comprehensive wellness programming as a means to reach students and parents. The original Switch trial that took place at 10 schools across two states showed efficacy to change 3rd and 4th grade students’ healthy-living behaviors  [umption) . Subsequumption) . As an aumption) ,14, the umption) ,16,17 thumption) . The schumption) . Supportumption) , which humption) ,19,20.Building upon the foundation of the SWITCH intervention for elementary school wellness and child health, the middle school version was recently developed, namely, SWITCH–Middle School (MS). Following the same implementation process framework , SWITCH–n = 3 with 110 students; 51 boys, 59 girls; 54 in 6th grade, 38 in 7th grade, 18 in 8th grade) or inexperienced schools . Middle schools that had counterpart elementary schools or fourth and/or fifth grades that participated in the SWITCH elementary school project in the year before (2017–2018) were deemed “experienced” schools, or otherwise as “inexperienced” schools.An effectiveness-implementation hybrid type 3 quasi-experimental design was followed to implement and evaluate the SWITCH–MS intervention . A type https://www.extension.iastate.edu/4h/) who facilitate county-level 4-H programming also attended the conference to learn about SWITCH–MS and then were tasked to work with the implementation teams from specific schools within their respective counties to set up SWITCH–MS goals and develop action plans. The 4-H stands for head, heart, hands, and health, and it is America’s largest youth development organization that empowers nearly six million young children with the skills to lead for a life time (https://4-h.org/about/what-is-4-h/). The Iowa State University Outreach and Extension 4-H was a partner of the SWITCH–MS project, who contributed to recruitment, training, and engaging of schools. They were considered key agents of change throughout the implementation of SWITCH–MS. A recent study has shown that the involvement of 4-H extension staff is important to change school capacity for wellness programing [www.iowaswitch.org). The schools completed baseline assessments before the spring 2019 semester started and then launched the 12-week SWITCH–MS programming. The intervention spanned from late January to early May (including interruptions of spring break and inclement weather). The schools completed post-test data collection in May 2019.The SWITCH–MS intervention program was expanded from the SWITCH elementary school version, an evidence-based intervention . Both inograming . ThroughThe whole-of-school implementation outcomes of the SWITCH–MS were self-reported by the key informants using a 21-item survey administered toward the end of implementation (week 12). Specifically, 9 questions assessed the implementation of best practices by the school team  and 12 questions assessed awareness of SWITCH–MS goals and staff engagement level (6 questions for each construct). These questions were answered on a 3-point scale. For example, a question that measures engagement in PE classes is phrased as: “How would your team rate the overall degree of engagement in PE?” Possible choices were: “1 = Low (little to no involvement)”, “2 = Moderate (some involvement but not enough to be a key role player in SWITCH)”, and “3 = High (Lots of involvement and playing a key role in SWITCH implementation)”.www.iowaswitch.org), to inform the classroom-based implementation outcome. Records of completed vs. not completed trackers were saved to the website. Completion rate among students per class was calculated to show the level of tracking behavior. The class-level tracking rate was aggregated by school and then by condition (inexperienced vs. experienced) for subsequent analysis. As documented in prior research, students’ behavioral tracking rate has been shown to be an important element of SWITCH implementation and functionality [In health education classrooms, students were asked to track their “Do” (at least 60 min per day), “View” , and “Chew” behaviors (5 or more servings of fruits and/or vegetables) each week, using the sliding bar on the project website  with a validated instrument for concurrent validity [Three healthy-living behaviors including “Do” (PA), “View” (screen-based sedentary behaviors), and “Chew” (fruits and vegetable consumption) were measured using the validated Youth Activity Profile (YAP) to determine the effectiveness of SWITCH–MS. YAP has 20 questions with 10 capturing “Do” (5 on PA at school and 5 on PA out of school) and 5 each capturing “View” and “Chew” behaviors. The questions were answered mainly on a 5-point scale. For example, a question that measures “Do” at school is phrased as: “How many days did you walk or bike to school? ”; “B. 1 day”; “C. 2 days”; “D. 3 days”; “E. 4‒5 days (most every day)”. Previous research reported that the 15 questions of YAP assessing PA and sedentary behavior could produce relatively accurate group-level estimates of PA and sedentary in comparison to objective measures ,28. For validity .Students’ healthy-living behaviors  were assessed using YAP before and after SWITCH–MS implementation. Students completed the questionnaire online at their school’s media center, or in their gymnasium, using laptop computers under the supervision of their teachers. The teachers received training through preparatory webinars on how to administer the questionnaire. Students were encouraged to ask questions when needed, and then completed the questionnaire independently. In addition, students were prompted by their health education classroom teachers to complete the “Do”, “View”, and “Chew” behavioral trackers each week during the 12 weeks of SWITCH–MS implementation. For the staff survey, the core school implementation team staff or key informants were asked to complete the survey online toward the end of the SWITCH–MS implementation (week 12). All data were automatically recorded and saved in the online server and then downloaded during summer 2019 for cleaning and processing.p value, and intra-class correlational (ICC) coefficient were reported as results from model 4 for each of the healthy-living behaviors. The sequential three-level linear mixed modeling described above was performed by following Heck et al.’s tutorials [Descriptive statistics  were obtained to evaluate the implementation of SWITCH–MS. First, for whole-of-school implementation evaluation, descriptive statistics for staff-reported implementation of best practices , awareness, and engagement were compared between inexperienced and experienced schools. For classroom implementation evaluation, a line chart was drawn to illustrate the weekly tracker completion rate for inexperienced vs. experienced schools across the 12 weeks. To determine the effectiveness of SWITCH–MS, a series of three-level linear mixed models  were specified, where student-level healthy-living behaviors were entered as dependent variables , with one dependent variable per model. Specifically, in model 1, we tested the null or no predictor model with randomly varying intercept at student and school–level. In model 2, we tested the fixed effects of time (pre vs. posttest), condition (inexperienced vs. experienced schools), and time–by–condition interaction with randomly varying intercepts at student and school levels. In model 3, we added gender and grade as student-level covariates to the model 2. In model 4, we added one of the four school–level covariates  to model 3. Socioeconomic status was not entered as a school level predictor due to its small variation between schools , “Do” out of school (p < 0.01), and “View” behaviors, favoring posttest behaviors (p = 0.02). These models showed a small clustering effect, with 1.8‒9.6% variances accounted for at the school level. Model 3 added gender and grade as student-level predictors to model 2, and model 4 added behavioral tracking, staff-reported implementation of best practice, awareness, and engagement, respectively. Compared to girls, boys showed significantly higher “Do” behaviors  but also higher “View” behavior (p < 0.01). By grade, 6th grade students displayed significantly higher “Do” behaviors, higher “Chew” behaviors, and lower “View” behaviors (p < 0.01) than 8th grade students; while 7th grade students also showed higher “Chew” behaviors than 8th grade students (p = 0.04). Behavior tracking, as a school-level covariate, only seemed to matter to “Do” out of school behavior (p = 0.03). To save space, Four sequential linear mixed models were conducted for each of the behavior outcome variables . The purpose of this study was to evaluate the implementation and effectiveness of the SWITCH–MS intervention, as a whole-of-school intervention. The implementation and effectiveness outcomes were compared between inexperienced vs. experienced schools, which showed similarities and differences that are theoretically and practically meaningful. The findings and their implications are discussed below.n = 3) showed slightly higher levels of engagement, but lower levels of best practice implementation in the lunchroom than the inexperienced schools (n = 3). Inferential statistics were not conducted on these implementation outcomes due to small sample size at the school level (n = 6). Furthermore, classroom-based implementation—as represented by weekly behavioral tracking rate for “Do”, “View”, and “Chew” behaviors—was better in the experienced schools during the first two weeks of implementation than that in the inexperienced schools, but the group differences were attenuated between weeks 3 and 12. As previously mentioned, behavioral tracking is an essential element of the SWITCH programing [The implementation evaluation of SWITCH–MS revealed some interesting findings about the implementation outcomes. First, the whole-of-school implementation of SWITCH–MS components were similar and commensurate between the two conditions of schools for utilizing best practices in PE, health classroom, and lunchroom; being cognizant of the SWITCH–MS goals at multiple levels (from teachers and lunchroom staff to parents’ involvement); and staying engaged in achieving these goals see . Howeverograming . Previouograming ,22, showThe effectiveness evaluation demonstrated that SWITCH–MS intervention was associated with significant temporal changes in most of the healthy-living behaviors  among middle school students at both inexperienced and experienced schools. Our three-level linear mixed models adjusted for student- and school-level predictors in order to accurately determine the intervention effect. The non-significant time-by-condition interaction effects for the healthy-living behaviors suggest that prior exposure to SWITCH in upper elementary school years did not matter when it comes to the effectiveness of SWITCH–MS on these behaviors. In other words, SWITCH–MS can render similar influence on promoting middle school students’ healthy-living behaviors (“Do” and “View”), which is not predicated on prior experiences. These results are consistent with the previous findings shown in the original elementary school Switch efficacy trial, verifying the potential of SWITCH programing in influencing school wellness and child health ,12,18.Taken together the implementation and effectiveness evaluation, this study provides evidence of SWITCH–MS, as an adolescent obesity prevention intervention. The findings verify the importance of following the SWITCH Implementation Process framework for implementation across environmental settings, attending preparatory webinars and in-person training, as well as the involvement of 4-H staff engagement ,26. MakiDespite the demonstrated implementation and effectiveness of the SWITCH–MS intervention, we acknowledge several limitations of this study. First, in this effectiveness-implementation hybrid type 3 design quasi–experimental study , we did This evaluation study has provided evidence for the implementation and effectiveness outcomes of SWITCH–MS, as a whole-of-school intervention, in changing middle school wellness and adolescents’ healthy-living behaviors. For implementation outcomes, the two groups of schools showed similar levels of self-reported best practices, awareness, and engagement. Classroom-based implementation, as represented by the behavioral-tracking practice, was also similar between the two groups of schools after the intervention was launched beyond two weeks. For effectiveness outcomes, students’ physical activity levels both during and out of school, and screen-time-based sedentary behaviors improved over the 12 weeks of intervention, regardless of treatment groups. Based on the evidence, we conclude that the implementation and effectiveness of SWITCH–MS are not predicated on students’ prior experience, although some implementation indicators  may favor the experienced schools than inexperienced schools. The findings shed light on future implementation and dissemination of the SWITCH–MS intervention in broader schools.