Effects of constant darkness on behaviour and physiology of male and female mice.

A healthy state of life suggests not only a disease-free condition but also normal psychological functioning and behaviour. To maintain a healthy life, the duration of light exposure is a crucial factor. Perturbation of the standard light-dark cycle (LD: 12 h light-12 h dark in mice) may result in brain, behavioural and physiological abnormalities. The current study determined the effects of 3 and 5 weeks of constant darkness (DD: 00 h light-24 h dark) on the behaviour, hormones, prefrontal cortex (PFC) and metabolome of male and female C57BL/6 J mice. We also studied 3 weeks of restoration in LD following 5 weeks of DD exposure. The results revealed that 3 weeks of DD affected male mice more than females, and 5 weeks of DD had a comparable impact on behaviour, hormones and the PFC of male and female mice. After restoration in LD, the DD-induced changes reverted to time-matched LD conditions in male and female mice. Furthermore, metabolome analysis corroborated male and female mice's behavioural and molecular kinetics. The present study laid the foundation for understanding how DD affects behaviour and the PFC as a function of (a) time and (b) sex and described the roles of stress and sex hormones, cytokines, neurotrophins and metabolic pathways.

Vancomycin-Induced Changes in Host Immunity and Behavior: Comparative Genomic and Metagenomic Analysis in C57BL/6 and BALB/c Mice.

BACKGROUND: The consequence of treatment with antibiotics on the gut microbiota can be destructive. The antibiotics, however, can be utilized to understand the role of gut microbiota on the host physiology. AIM: Earlier, we reported the efficacy of vancomycin in gut microbiota perturbation. We continued to understand the effect of restoration kinetics of perturbed gut microbiota on the immunity and behavior of Th1 (C57BL/6)- and Th2 (BALB/c)-biased mice. METHODS: We studied restoration kinetics of the gut microbiota for two months following the withdrawal of vancomycin treatment in both mice strains. We analyzed cecal microbiome composition, different behavioral assays, and expression of select genes associated with stress and barrier function in gut and brain. RESULTS: Metagenomic analysis of gut microbiota revealed that the treatment with vancomycin caused a significant decrease in the relative abundance of Firmicutes and Bacteroidetes phyla with a time-dependent increase in Proteobacteria and Verrucomicrobia phyla. Maximum restoration (> 70%) of gut microbiota happened by the 15th day of withdrawal of vancomycin. BALB/c mice showed a more efficient restoration of gut microbiota compared to C57BL/6 mice. We established the correlation patterns of gut microbiota alteration and its effect on (a) the behavior of mice, (b) expression of key brain molecules, and (c) immunity-related genes. CONCLUSIONS: The results revealed that the gut microbiome profiling, behavior, and immune responses varied significantly between Th1- and Th2-biased mice. By withdrawing the treatment with vancomycin of major gut microbes, important physiological and behavioral changes of both mice strains returned to the normal (untreated control) level.

Vaginal microbiota of asymptomatic bacterial vaginosis and vulvovaginal candidiasis: Are they different from normal microbiota?

Vaginal microbiota contributes in maintaining and protecting the urogenital niche from infections and their sequelae. Despite extensive research, microbiome studies have often ignored asymptomatic bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC). The present study aimed to explore the cultivable vaginal bacterial and mycological communities in women asymptomatic for BV and VVC using multiplex PCR and species-specific PCR. Vaginal swabs collected from 199 participants asymptomatic for urogenital infections, scored by Nugent criteria indicated 73.9% had normal microbiota, 11.6% intermediate and 14.5% BV. The most frequent Lactobacillus species in normal women were L. iners (69.4%), L. crispatus (24.5%), L. reuteri (20.4%). Women with BV colonized L. iners (62.1%); L. rhamnosus (41.4%); L. salivarius (13.7%) and L. reuteri (7.2%). Furthermore, L. crispatus was associated with normal microbiota, whereas L. iners was a frequent member of normal and dysbiotic microbiota. Lactobacillus abundance and species richness reduced in asymptomatic BV. Also L. crispatus, L. fermentum, L. acidophilus and L. delbruckii were absent in these women. L. iners significantly co-existed with other Lactobacillus species, indicating its failure in independently maintaining the healthy vaginal niche. Of 30.4% women detected with Candida, 72.1% constituted non-albicans Candida. Predominance of C. albicans increased from 18.4% in healthy to 60% in women with asymptomatic BV; whereas distribution of BV related bacteria did not vary across the groups. Heterogeneous population of lactobacilli in 80.8% of normal women calls attention towards cumulative effects of these species in safeguarding the vaginal microenvironment. Since the microbiota of asymptomatic BV was different from healthy, screening and management could be encouraged to avoid further complications of infections.

Comparative efficacy analysis of anti-microbial peptides, LL-37 and indolicidin upon conjugation with CNT, in human monocytes.

BACKGROUND: Antimicrobial peptides (AMPs) have the potential to serve as an alternative to antibiotic. AMPs usually exert bactericidal activity via direct killing of microbial pathogens. Reports have proposed that by harnessing innate immune activation, AMPs can regulate pathogen invasion and may control infection. It has been reported that AMPs could be utilized to activate the innate mucosal immune response in order to eliminate pathogenic infections. This way of controlling pathogen infection, by activating host immunity, confers the potential to the select AMPs to alleviate the problem of antibiotic resistance. Among various AMPs tested LL-37 and indolicidin, showed promise to be potential candidates for eliciting enhanced host innate immune responses. LL-37 and indolicidin had exhibited substantial innate immune activation in both human and murine macrophages. Dosage for each of the AMPs, however, was high with adverse side effects. RESULTS: In this study, we reported that upon conjugation with carbon nanotubes (CNT), each AMP remained biologically functional at a concentration that was 1000-fold less than the dosage required for free AMP to remain active in the cells. CONCLUSIONS: Current study also revealed that while indolicidin induced signalling events mediated through the TNFRSF1A pathway in THP1 cells, followed by activation of NFκB and c-JUN pathways, treatment of cells with LL-37 induced signalling events by activating IL1R, with subsequent activation of NFκB and NFAT2. Thp1 cells, primed with CNT conjugated LL-37 or indolicidin, are protected against Salmonella typhimurium infection at 16 h post challenge.

Antibiotic induced adipose tissue browning in C57BL/6 mice: An association with the metabolic profile and the gut microbiota.

AIMS: The use of antibiotics affects health. The gut microbial dysbiosis by antibiotics is thought to be an essential pathway to influence health. It is important to have optimized energy utilization, in which adipose tissues (AT) play crucial roles in maintaining health. Adipocytes regulate the balance between energy expenditure and storage. While it is known that white adipose tissue (WAT) stores energy and brown adipose tissue (BAT) produces energy by thermogenesis, the role of an intermediate AT plays an important role in balancing host internal energy. In the current study, we tried to understand how treating an antibiotic cocktail transforms WAT into BAT or, more precisely, into beige adipose tissue (BeAT). METHODS: Since antibiotic treatment perturbs the host microbiota, we wanted to understand the role of gut microbial dysbiosis in transforming WAT into BeAT in C57BL/6 mice. We further correlated the metabolic profile at the systemic level with this BeAT transformation and gut microbiota profile. KEY FINDINGS: In the present study, we have reported that the antibiotic cocktail treatment increases the Proteobacteria and Actinobacteria while reducing the Bacteroidetes phylum. We observed that prolonged antibiotic treatment could induce the formation of BeAT in the inguinal and perigonadal AT. The correlation analysis showed an association between the gut microbiota phyla, beige adipose tissue markers, and serum metabolites. SIGNIFICANCE: Our study revealed that the gut microbiota has a significant role in regulating the metabolic health of the host via microbiota-adipose axis communication.

Postnatal intestinal mucosa and gut microbial composition develop hand in hand: A mouse study.

BACKGROUND: During the early postnatal life, gut microbiota development experiences dynamic changes in their structural and functional composition. The postnatal period is the critical window to develop a host defense mechanism. The maturation of intestinal mucosal barrier integrity is one of the essential defense mechanisms to prevent the entry of pathogens. However, the co-development of intestinal microbial colonization, formation of barrier integrity, and intestinal epithelial cell layer is not entirely understood. METHODS: We studied the gut microbial composition and diversity using 16S rRNA marker gene-based sequencing in mice to understand postnatal age-dependent association kinetics between gut microbial and intestinal development. Next, we assessed the intestinal development by in vivo gut permeability assay, mRNA gene expression of different tight junction proteins and intestinal epithelial cell markers, goblet cells population, villus length, and cecal IgA quantification. RESULTS: Our results showed a significant shift in gut microbial structural and functional composition from postnatal day 14 onwards with early life Proteobacteria abundance. Relative abundance of Verrucomicrobia was maximum at postnatal day 14 and showed a gradual decrease over time. We also observed an age-dependent biphasic pattern in barrier integrity improvement and differentiation of intestinal epithelial cells (IECs). A significant improvement in barrier integrity between days 1 and 7 showed the host factor contribution, while that beyond day 14 revealed an association with changes in microbiota composition. Our temporal correlation analysis associated Bacteroidetes phylum with the mucosal barrier formation during postnatal development. CONCLUSIONS: The present study revealed the importance and interplay of host factors and the microbiome in gut development and intestinal mucosal homeostasis.

A comparative analysis of gut microbial dysbiosis by select antibiotics and DSS to understand the effects of perturbation on the host immunity and metabolism.

AIMS: Balanced gut microbial composition of the host plays a crucial role in maintaining harmony among various physiological processes to maintain physiological homeostasis. Immunity and metabolism are the two physiologies mainly controlled by the gut microbiota. Reports suggested that gut microbial composition and diversity alteration are the leading causes of the host's healthy homeostasis alteration or a diseased state. The extent of gut perturbation depends on the perturbing agents' strength, chemical nature, and mode of action. In the current report, we have studied the effects of different perturbing agents on gut microbial dysbiosis and its impact on host immunity and metabolism. MATERIALS AND METHODS: We studied the perturbation of gut microbial composition and diversity using next-generation sequencing and further investigated the changes in host immune and metabolic responses. KEY FINDINGS: Enrichment or abolition of a particular phylum or genus depended on the perturbing agents. In the current study, treatment with neomycin yielded an increase in the Bacteroidetes phylum. Vancomycin treatment caused a significant rise in Verrucomicrobia and Proteobacteria phyla. The treatment with AVNM and DSS caused a substantial increase in the Proteobacteria phylum. The gut microbial diversity was also lowest in AVNM treated group. The altered gut microbial composition ultimately altered the immune responses at localized and systemic levels of the host. Gut dysbiosis also changed the systemic level of SCFAs. SIGNIFICANCE: This study will help us understand how the enrichment of a particular phylum and genus maintains the host's immune responses and metabolism.

Postnatal 14D is the Key Window for Mice Intestinal Development- An Insight from Age-Dependent Antibiotic-Mediated Gut Microbial Dysbiosis Study.

The postnatal period is one of the critical windows for the structure-function development of the gastrointestinal tract and associated mucosal immunity. Along with other constituent members, recent studies suggest the contribution of gut microbiota in maintaining host health, immunity, and development. Although the gut microbiota's role in maintaining barrier integrity is known, its function in early life development still needs to be better understood. To understand the details of gut microbiota's effects on intestinal integrity, epithelium development, and immune profile, the route of antibiotic-mediated perturbation is taken. Mice on days 7(P7D), 14(P14D), 21(P21D) and 28(P28D) are sacrificed and 16S rRNA metagenomic analysis is performed. The barrier integrity, tight junction proteins (TJPs) expression, intestinal epithelial cell (IEC) markers, and inflammatory cytokines are analyzed. Results reveal a postnatal age-related impact of gut microbiota perturbation, with a gradual increase in the relative abundance of Proteobacteria and a reduction in Bacteroidetes and Firmicutes. Significant barrier integrity disruption, reduced TJPs and IECs marker expression, and increased systemic inflammation at P14D of AVNM-treated mice are found. Moreover, the microbiota transplantation shows recolonization of Verrucomicrobia, proving a causal role in barrier functions. The investigation reveals P14D as a critical period for neonatal intestinal development, regulated by specific microbiota composition.

A Systematic Review on the Association between Obesity and Mood Disorders and the Role of Gut Microbiota.

Obesity is a complex health condition that increases the susceptibility to developing cardiovascular diseases, diabetes, and numerous other metabolic health issues. The effect of obesity is not just limited to the conditions mentioned above; it is also seen to have a profound impact on the patient's mental state, leading to the onset of various mental disorders, particularly mood disorders. Therefore, it is necessary to understand the mechanism underlying the crosstalk between obesity and mental disorders. The gut microbiota is vital in regulating and maintaining host physiology, including metabolism and neuronal circuits. Because of this newly developed understanding of gut microbiota role, here we evaluated the published diverse information to summarize the achievement in the field. In this review, we gave an overview of the association between obesity, mental disorders, and the role of gut microbiota there. Further new guidelines and experimental tools are necessary to understand the microbial contribution to regulate a balanced healthy life.

Sex and Time: Important Variables for Understanding the Impact of Constant Darkness on Behavior, Brain, and Physiology.

The circadian clock can coordinate, regulate and predict physiology and behavior in response to the standard light-dark (LD: 12 h light and 12 h dark) cycle. If we alter the LD cycle by exposing mice to constant darkness (DD: 00 h light and 24 h dark), it can perturb behavior, the brain, and associated physiological parameters. The length of DD exposure and the sex of experimental animals are crucial variables that could alter the impact of DD on the brain, behavior, and physiology, which have not yet been explored. We exposed mice to DD for three and five weeks and studied their impact on (1) behavior, (2) hormones, (3) the prefrontal cortex, and (4) metabolites in male and female mice. We also studied the effect of three weeks of standard light-dark cycle restoration after five weeks of DD on the parameters mentioned above. We found that DD exposure was associated with anxiety-like behavior, increased corticosterone and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß), downregulated neurotrophins (BDNF and NGF), and altered metabolites profile in a duration of DD exposure and sex-dependent manner. Females showed a more robust adaptation than males under DD exposure. Three weeks of restoration was adequate to establish homeostasis in both sexes. To the best of our knowledge, this study is the first of its kind to look at how DD exposure impacts physiology and behavior as a function of sex- and time. These findings would have translational value and may help in establishing sex-specific interventions for addressing DD-related psychological issues.

Stress significantly increases mortality following a secondary bacterial respiratory infection.

A variety of mechanisms contribute to the viral-bacterial synergy which results in fatal secondary bacterial respiratory infections. Epidemiological investigations have implicated physical and psychological stressors as factors contributing to the incidence and severity of respiratory infections and psychological stress alters host responses to experimental viral respiratory infections. The effect of stress on secondary bacterial respiratory infections has not, however, been investigated. A natural model of secondary bacterial respiratory infection in naive calves was used to determine if weaning and maternal separation (WMS) significantly altered mortality when compared to calves pre-adapted (PA) to this psychological stressor. Following weaning, calves were challenged with Mannheimia haemolytica four days after a primary bovine herpesvirus-1 (BHV-1) respiratory infection. Mortality doubled in WMS calves when compared to calves pre-adapted to weaning for two weeks prior to the viral respiratory infection. Similar results were observed in two independent experiments and fatal viral-bacterial synergy did not extend beyond the time of viral shedding. Virus shedding did not differ significantly between treatment groups but innate immune responses during viral infection, including IFN-γ secretion, the acute-phase inflammatory response, CD14 expression, and LPS-induced TNFα production, were significantly greater in WMS versus PA calves. These observations demonstrate that weaning and maternal separation at the time of a primary BHV-1 respiratory infection increased innate immune responses that correlated significantly with mortality following a secondary bacterial respiratory infection.

Cost Effective Method for gDNA Isolation from the Cecal Content and High Yield Procedure for RNA Isolation from the Colonic Tissue of Mice.

Microbiome studies are quickly gaining momentum. Since most of the resident microbes (consisting of bacteria, fungi, and viruses) are difficult to culture, sequencing the microbial genome is the method of choice to characterize them. It is therefore important to have efficient methodology for gDNA isolation of gut microbes. Mouse models are widely used to understand human disease etiology while avoiding human ethics-related complications. However, the widely used kit-based methods are costly, and sometimes yields (in terms of quality and quantity) are sub-optimal. To overcome this problem, we developed a straightforward, standardized DNA isolation procedure from mouse cecal content for further microbiome-related studies. The reagents we used to standardize the procedure are readily available even in a not-so-well-equipped laboratory, and the reagents are not expensive. The yield and quality of the DNA are also better than those obtained by the readily available kit-based methods. Additionally, we modified the kit-based method of RNA isolation from the colon tissue sample of the mouse for better yield. Churning the tissue with liquid nitrogen at the beginning of the procedure improves RNA quality and quantity. Graphical abstract.

Effects of starch-rich or fat-rich diets on metabolism, adiposity, and glycemia in immune-biased, C57BL/6 and BALB/c mice.

Diet maintains health by regulating host metabolism and immunity. The results revealed the consequence of starch-, unsaturated fat-, and saturated fat-rich diets on differentially immune-biased mice C57BL/6 and BALB/c. Time-course of various diets on differentially immune-biased mice revealed that starch-rich and unsaturated fat-rich diets reduced insulin resistance (IR) and visceral adiposity in BALB/c mice while a saturated fat-rich diet enhanced both parameters. In C57BL/6 mice, a fat-rich diet enhanced IR with time while visceral adiposity remained unchanged. Eight weeks' consumption of saturated fat-rich diet induced highest visceral adiposity in C57BL/6 mice, while the same diet resulted in the maximum IR in BALB/c mice. The current report presented a detailed metabolomic analysis of diets and evaluated differential index of each treatment for each mouse strain using a vector analysis of the multivariate linear discriminant data. The outcome identified metabolites that affected lipid and glucose metabolism to establish the inter-strain physiological differences.

Microbiota transplantation from younger to older mice could restore lost immunity to effectively clear salmonella infection in Th2-biased BALB/c mice.

AIMS: The composition, overtly abundance, and diversity of gut microbiota, play a significant role in maintaining physiological homeostasis with age. Reports revealed that the gut microbial profile might be correlated with immunity and metabolism. It is, therefore, tantamount to know if an older individual can achieve the immunity and metabolic profile of a younger individual by receiving the gut microbiome of a younger individual. In the current report, we have studied the effects of cecal microbiota transplantation (CMT) from younger to older mice. MATERIALS AND METHODS: In this study, older BALB/c mice (23 weeks) received CMT from younger BALB/c mice (3 weeks). KEY FINDINGS: CMT recipient mice showed altered expressions of immune and tight junction protein genes in the colon of mice, while the non-CMT recipient mice did not. Older mice were treated with AVNM to make them compatible with CMT. Further data from metabolite studies revealed that AVNM treatment mainly affected the aromatic amino acid biosynthesis pathway while CMT mostly affected the metabolism of different carbohydrates. We repeated the analysis in C57BL/6 mice without any significant effects of CMT. SIGNIFICANCE: Results revealed that mice who received CMT showed more efficient restoration of gut microbiota than non-CMT recipient mice. CMT caused the alleviation of Salmonella infection and efficient recovery of the cecal index in the mice following antibiotics treatment.

Association between liver enzymes and metabolic syndrome in Canadian adults: results from the Canadian health measures survey - cycles 3 &4.

Background: The relationship between liver enzymes and Metabolic Syndrome (MetS) in different populations, including Canadians, is not consistent and well understood. We used the Canadian Health Measures Survey data (Cycles 3 and 4) to examine the cross-sectional relationships between select liver biomarkers and MetS in the adult Canadian population. The biomarkers selected were gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST), and alkaline phosphatase (ALKP). Methods: Fasting blood samples (FBS) were collected from adults above the age of 20 years for Cycle 3 and Cycle 4 (n = 3003). MetS was diagnosed if the subjects had three or more risk determinants according to the Joint Interim Statement criteria. Primary risk factors included quartile cut-offs for each of the biomarkers ALKP, AST, GGT for males and females separately. A multivariable logistic regression technique based on a maximum likelihood approach was used to evaluate the association between quartiles of ALKP, AST, and GGT, other individual and contextual factors, and the prevalence of MetS. Results: MetS was prevalent in 32.3% of subjects. BMI was an effect modifier in the relationship between GGT and MetS prevalence, while sex was an effect modifier in the relationship between ALKP and MetS prevalence; and age was an effect modifier in the relationship between AST and MetS prevalence. Conclusions: Since the mechanisms to underpin the associations between the liver enzymes activity and MetS are unknown, further epidemiologic investigations using longitudinal designs are necessary to understand these associations.

Differential colitis susceptibility of Th1- and Th2-biased mice: A multi-omics approach.

The health and economic burden of colitis is increasing globally. Understanding the role of host genetics and metagenomics is essential to establish the molecular basis of colitis pathogenesis. In the present study, we have used a common composite dose of DSS to compare the differential disease severity response in C57BL/6 (Th1 biased) and BALB/c (Th2 biased) mice with zero mortality rates. We employed multi-omics approaches and developed a newer vector analysis approach to understand the molecular basis of the disease pathogenesis. In the current report, comparative transcriptomics, metabonomics, and metagenomics analyses revealed that the Th1 background of C57BL/6 induced intense inflammatory responses throughout the treatment period. On the contrary, the Th2 background of BALB/c resisted severe inflammatory responses by modulating the host's inflammatory, metabolic, and gut microbial profile. The multi-omics approach also helped us discover some unique metabolic and microbial markers associated with the disease severity. These biomarkers could be used in diagnostics.

Impact of sodium glucose cotransporter-2 inhibitors on liver steatosis/fibrosis/inflammation and redox balance in non-alcoholic fatty liver disease.

BACKGROUND: Sodium glucose cotransporter-2 inhibitors (SGLT2-I) are the most recently approved drugs for type 2 diabetes (T2D). Recent clinical trials of these compounds reported beneficial cardiovascular (CV) and renal outcomes. A major cause of vascular dysfunction and CV disease in diabetes is hyperglycemia associated with inflammation and oxidative stress. Pre-clinical studies demonstrated that SGLT2-I reduce glucotoxicity and promote anti-inflammatory effects by lowering oxidative stress. AIM: To investigate the effects of SGLT2-I on markers of oxidative stress, inflammation, liver steatosis, and fibrosis in patients of T2D with non-alcoholic fatty liver disease (NAFLD). METHODS: We referred fifty-two consecutive outpatients treated with metformin monotherapy and exhibiting poor glycemic control to our centre. We introduced the outpatients to an SGLT2-I (dapagliflozin, empagliflozin, or canagliflozin; n = 26) or a different hypoglycemic drug [other glucose-lowering drugs (OTHER), n = 26]. We evaluated circulating interleukins and serum hydroxynonenal (HNE)- or malondialdehyde (MDA)-protein adducts, fatty liver index (FLI), NAFLD fibrosis score, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio, AST-to-platelet-ratio index (APRI), and fibrosis-4 on the day before (T0) and following treatment for six months (T1). We also performed transient elastography at T0 and T1. RESULTS: Add-on therapy resulted in improved glycemic control and reduced fasting blood glucose in both groups. Of note, following treatment for six months, a reduction of FLI and APRI, as well as of the FibroScan result, was reported in patients treated with SGLT2-I, but not in the OTHER group; furthermore, in the SGLT2-I group, we reported lower circulating levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor, vascular endothelial growth factor, and monocyte chemoattractant protein-1, and higher levels of IL-4 and IL-10. We did not observe any modification in circulating interleukins in the OTHER group. Finally, serum HNE- and MDA-protein adducts decreased significantly in SGLT2-I rather than OTHER patients and correlated with liver steatosis and fibrosis scores. CONCLUSION: The present data indicate that treatment with SGLT2-I in patients with T2D and NAFLD is associated with improvement of liver steatosis and fibrosis markers and circulating pro-inflammatory and redox status, more than optimizing glycemic control.

Effects of two potential probiotic Lactobacillus bacteria on adipogenesis in vitro.

AIMS: Overweight is a major global health problem. Various methodologies to get rid of the extra fat are available, but usually, those are associated with adverse side effects. Probiotics, on the contrary, seem to have the potential to help reduce fat accumulation without much apparent adversity. In this study, we have evaluated a pair of well-documented probiotics for their anti-obesogenic effects. MAIN METHODS: We used strains of Lactobacillus acidophilus (LA) and a cocktail (LDB-ST) of Lactobacillus delbruckei sp. bulgaricus (LDB) and Streptococcus thermophilus (ST) in this study. The murine pre-adipocyte cell line 3T3-L1 was terminally differentiated to matured adipocytes to use as a model to evaluate the bacteria's anti-obesogenic effects. The optimal dose for treatment of both the probiotics was determined using a cell viability assay. We assessed the probiotic internalization potential of differentiated 3T3-L1 cells by flow cytometry, fluorescence microscopy, and cell lysis method. We determined the lipolytic and anti-adipogenic potential of probiotics by intracellular lipid staining, spectrophotometry, and gene expression analysis. KEY FINDINGS: Both probiotics were effective lipolytic agents as revealed by reducing cellular lipids and down-regulation of mammalian adipogenesis marker genes in terminally differentiated 3T3-L1 cells. SIGNIFICANCE: Previous studies from our group had proven the immune-modulatory properties of these probiotics on an immune-biased mouse model. The present study demonstrates LA and LDB-ST to be effective against adipogenesis. Further in vivo studies will be conducted to strengthen this claim.

Predictive biomarkers of cardiovascular disease in adult Canadian population.

BACKGROUND: Elevated levels of the enzymes gamma-glutamyltransferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and C-reactive protein (CRP) have been shown to be associated with increased risk of cardiovascular disease (CVD). Objective: To assess cross-sectional relationships between biomarkers GGT, ALT, AST, ALP and CVD in adult Canadian population. METHODS: The Canadian Health Measures Surveys (CHMSs) are a series of cross-sectional national surveys and collect information on indicators of general health and wellness of Canadians. The CHMS has four components. We used data from the first three components (for Study participants ≥ 20 years) from CHMS cycles 1 through 5. RESULTS: Multivariable logistic regression revealed: immigration status [Odds ratio (OR)(95% Confidence Interval (95% CI)) = 0.67 (0.53-0.85), reference category (RC)-no-immigrant] education [1.38(1.10-1.75), RC- > secondary education]; smoking status [ex-smokers: 1.16(0.89-1.51); current smokers: 1.41(0.98-2.05), RC-non-smoker]; and income [middle income: 0.69(0.43-1.10); high income: 0.49(0.29-0.83); RC-lower income] were significantly associated with CVD prevalence. CONCLUSION: The relationship of GGT with CVD prevalence changed among age groups and body mass index categories; was different for males and females; and diabetes was an effect modifier in the relationship between AST and CVD prevalence. Socio-economic factors were significantly associated with CVD prevalence.

Secretory clusterin promotes oral cancer cell survival via inhibiting apoptosis by activation of autophagy in AMPK/mTOR/ULK1 dependent pathway.

AIMS: Secretory clusterin (sCLU) plays an important role in tumor development and cancer progression. However, the molecular mechanisms and physiological functions of sCLU in oral cancer is unclear. We examined the impact of sCLU-mediated autophagy in cell survival and apoptosis inhibition in oral cancer. MAIN METHODS: Immunohistochemical analysis was performed to analyze protein expression in patient samples. Autophagy and mitophagy was studied by immunofluorescence microscopy and Western blot. The gain and loss of function was studied by overexpression of plasmid and siRNA approaches respectively. Cellular protection against nutrient starvation and therapeutic stress by sCLU was studied by cell viability, caspase assay and meta-analysis. KEY FINDINGS: The data from oral cancer patients showed that the expression levels of sCLU, ATG14, ULK1, and PARKIN increased in grade-wise manners. Interestingly, sCLU overexpression promoted autophagy through AMPK/Akt/mTOR signaling pathway leading to cell survival and protection from long exposure serum starvation induced-apoptosis. Additionally, sCLU was demonstrated to interact with ULK1 and inhibition of ULK1 activity by SBI206965 was found to abolish sCLU-induced autophagy indicating critical role of ULK1 in induction of autophagy. Furthermore, sCLU was observed to promote expression of mitophagy-associated proteins in serum starvation conditions to protect cells from nutrient deprivation. The meta-analysis elucidated that high CLU expression is associated with therapy resistance in cancer and we demonstrated that sCLU-mediated mitophagy was revealed to inhibit cell death by cisplatin. SIGNIFICANCE: The present investigation has highlighted the probable implications of the clusterin-induced autophagy in cell survival and inhibition of apoptosis in oral cancer.