Intraduodenal Fecal Microbiota Transplantation Ameliorates Gut Atrophy and Cholestasis in a Novel Parenteral Nutrition Piglet Model.

BACKGROUND: Total parenteral nutrition (TPN) provides lifesaving nutritional support intravenously; however it is associated with significant side effects. Given gut microbial alterations noted with TPN, we hypothesized that transferring fecal microbiota from healthy controls would restore gut-systemic signaling in TPN and mitigate injury. METHODS: Using our novel ambulatory model (US Patent: US 63/136,165), 31 piglets were randomly allocated to enteral nutrition (EN), TPN only, TPN + antibiotics (TPN-A) or TPN + intraduodenal fecal microbiota transplant (TPN-FMT) for 14 days. Gut, liver, and serum were assessed through histology, biochemistry, and qPCR. Stool samples underwent 16s rRNA sequencing. PERMANOVA, Jaccard and Bray-Curtis metrics were performed. RESULTS: Significant bilirubin elevation in TPN and TPN-A vs EN (p<0.0001) was prevented with FMT. IFN-G, TNF-alpha, IL-beta, IL-8 and LPS were significantly higher in TPN (p=0.009/0.001/0.043/0.011/<0.0001), with preservation upon FMT. Significant gut-atrophy by villous/crypt ratio in TPN (p<0.0001) and TPN-A (p=0.0001) vs EN was prevented by FMT (p=0.426 vs EN). Microbiota profiles using Principal Coordinate Analysis demonstrated significant FMT and EN overlap, with the largest separation in TPN-A followed by TPN, driven primarily by Firmicutes and Fusobacteria. TPN altered gut barrier was preserved upon FMT. Upregulated CYP7A1 and BSEP in TPN and TPN-A, and downregulatedFGFR4, EGF, FXR and TGR5 vs EN was prevented by FMT. CONCLUSION: This study provides novel evidence of prevention of gut atrophy, liver injury and microbial dysbiosis with intraduodenal FMT, challenging current paradigms into TPN injury mechanisms and underscores importance of gut microbes as prime targets for therapeutics and drug discovery.

The impact of national income and vaccine hesitancy on country-level COVID-19 vaccine uptake.

BACKGROUND: The rapid development and rollout of COVID-19 vaccines helped reduce the pandemic's mortality burden. The vaccine rollout, however, has been uneven; it is well known that vaccination rates tend to be lower in lower income countries. Vaccine uptake, however, ultimately depends on the willingness of individuals to get vaccinated. If vaccine confidence is low, then uptake will be low, regardless of country income level. We investigated the impact on country-level COVID-19 vaccination rates of both national income and vaccine hesitancy. METHODS: We estimated a linear regression model of COVID-19 vaccine uptake across 145 countries; this cross-sectional model was estimated at each of four time points: 6, 12, 18, and 24 months after the onset of global vaccine distribution. Vaccine uptake reflects the percentage of the population that had completed their primary vaccination series at the time point. Covariates include per capita GDP, an estimate of the percentage of country residents who strongly disagreed that vaccines are safe, and a variety of control variables. Next, we estimated these models of vaccine uptake by country income (countries below, and above the international median per capita GDP) to examine whether the impact of vaccine hesitancy varies by country income. RESULTS: We find that GDP per capita has a pronounced impact on vaccine uptake at 6 months after global rollout. After controlling for other factors, there was a 22 percentage point difference in vaccination rates between the top 20% and the bottom 20% of countries ranked by per capita GDP; this difference grew to 38% by 12 months. The deleterious impact of distrust of vaccine safety on vaccine uptake became apparent by 12 months and then increased over time. At 24 months, there was a 17% difference in vaccination rates between the top 20% and the bottom 20% of countries ranked by distrust. The income stratified models reveal that the deleterious impact of vaccine distrust on vaccine uptake at 12 and 24 months is particularly evident in lower income countries. CONCLUSIONS: Our study highlights the important role of both national income and vaccine hesitancy in determining COVID-19 vaccine uptake globally. There is a need to increase the supply and distribution of pandemic vaccines to lower-income countries, and to take measures to improve vaccine confidence in these countries.

Gut Microbiota to Microglia: Microbiome Influences Neurodevelopment in the CNS.

The brain is traditionally viewed as an immunologically privileged site; however, there are known to be multiple resident immune cells that influence the CNS environment and are reactive to extra-CNS signaling. Microglia are an important component of this system, which influences early neurodevelopment in addition to modulating inflammation and regenerative responses to injury and infection. Microglia are influenced by gut microbiome-derived metabolites, both as part of their normal function and potentially in pathological patterns that may induce neurodevelopmental disabilities or behavioral changes. This review aims to summarize the mounting evidence indicating that, not only is the Gut-Brain axis mediated by metabolites and microglia throughout an organism's lifetime, but it is also influenced prenatally by maternal microbiome and diet, which holds implications for both early neuropathology and neurodevelopment.

Gut Microbiota Modulation of Short Bowel Syndrome and the Gut-Brain Axis.

Short bowel syndrome (SBS) is a condition that results from a reduction in the length of the intestine or its functional capacity. SBS patients can have significant side effects and complications, the etiology of which remains ill-defined. Thus, facilitating intestinal adaptation in SBS remains a major research focus. Emerging data supports the role of the gut microbiome in modulating disease progression. There has been ongoing debate on defining a "healthy" gut microbiome, which has led to many studies analyzing the bacterial composition and shifts that occur in gastrointestinal disease states such as SBS and the resulting systemic effects. In SBS, it has also been found that microbial shifts are highly variable and dependent on many factors, including the anatomical location of bowel resection, length, and structure of the remnant bowel, as well as associated small intestinal bacterial overgrowth (SIBO). Recent data also notes a bidirectional communication that occurs between enteric and central nervous systems called the gut-brain axis (GBA), which is regulated by the gut microbes. Ultimately, the role of the microbiome in disease states such as SBS have many clinical implications and warrant further investigation. The focus of this review is to characterize the role of the gut microbiota in short bowel syndrome and its impact on the GBA, as well as the therapeutic potential of altering the microbiome.

Intracellular Zinc Trafficking during Crotalus atrox Venom Wound Development.

In this study, we examined zinc trafficking in human umbilical vein endothelial cells (HUVEC) stimulated with Crotalus atrox (CA venom) snake venom. We utilized MTS cytotoxicity assays to monitor the cytotoxic range of CA venom. HUVEC monolayers stimulated with 10 µg/mL CA venom for 3 h displayed cellular retraction, which coincided with 53.0 ± 6.5 percent viability. In contrast, venom concentrations of 100 µg/mL produced a complete disruption of cellular adherence and viability decreased to 36.6 ± 1.0. The zinc probe Fluozin-3AM was used to detect intracellular zinc in non-stimulated controls, HUVEC stimulated with 10 µg/mL CA venom or HUVEC preincubated with TPEN for 2 h then stimulated with 10 µg/mL CA venom. Fluorescent intensity analysis returned values of 1434.3 ± 197.4 for CA venom demonstrating an increase of about two orders of magnitude in labile zinc compared to non-stimulated controls. Endothelial response to CA venom induced a 96.1 ± 3.0- and 4.4 ± 0.41-fold increase in metallothionein 1X (MT1X) and metallothionein 2A (MT2A) gene expression. Zinc chelation during CA venom stimulation significantly increased cell viability, suggesting that the maintenance of zinc homeostasis during envenomation injury improves cell survival.

Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice.

BACKGROUND: Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified. METHODS: To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior. RESULTS: Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits. CONCLUSIONS: This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.

An in vivo Cell-Based Delivery Platform for Zinc Finger Artificial Transcription Factors in Pre-clinical Animal Models.

Zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas9 therapies to regulate gene expression are becoming viable strategies to treat genetic disorders, although effective in vivo delivery systems for these proteins remain a major translational hurdle. We describe the use of a mesenchymal stem/stromal cell (MSC)-based delivery system for the secretion of a ZF protein (ZF-MSC) in transgenic mouse models and young rhesus monkeys. Secreted ZF protein from mouse ZF-MSC was detectable within the hippocampus 1 week following intracranial or cisterna magna (CM) injection. Secreted ZF activated the imprinted paternal Ube3a in a transgenic reporter mouse and ameliorated motor deficits in a Ube3a deletion Angelman Syndrome (AS) mouse. Intrathecally administered autologous rhesus MSCs were well-tolerated for 3 weeks following administration and secreted ZF protein was detectable within the cerebrospinal fluid (CSF), midbrain, and spinal cord. This approach is less invasive when compared to direct intracranial injection which requires a surgical procedure.

Artificial escape from XCI by DNA methylation editing of the CDKL5 gene.

A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.

The iNs and Outs of Direct Reprogramming to Induced Neurons.

Understanding of cell-type specific transcription factors has promoted progress in methods for cellular reprogramming, such as directly reprogramming somatic cells to induced neurons (iN). Methods for direct reprogramming require neuronal-fate determining gene activation via neuron-specific microRNAs, chemical modulation of key neuronal signaling pathways or overexpression via viral vectors, with some reprogramming strategies requiring a combination of these methods to induce the neuronal-cell fate. These methods have been employed in a multitude of cell types, including fibroblasts, hepatocytes, peripheral blood mononuclear, and T cells. The ability to create iN from skin biopsies and blood samples coupled with recent advancements in artificially inducing age- and disease-associated phenotypes are accelerating the development of disease models for late-onset neurodegenerative disorders. Here, we review how activation of the neuronal transcriptome alters the epigenetic landscape of the donor cell to facilitate reprogramming to neurons. We also discuss the advantages of using DNA binding domains such as CRISPR/dCas9 to overcome epigenetic barriers to induce neuronal-cell fate by activating endogenous neuronal cell-fate determining genes.

Age-Related Improvements in Peak Cardiorespiratory Fitness among Coronary Heart Disease Patients Following Cardiac Rehabilitation.

While cardiorespiratory fitness (VO2peak) can be improved with exercise and training, it is unclear whether older age is associated with an attenuated VO2peak improvement among patients with coronary artery disease (CAD) who complete a cardiac rehabilitation (CR) program. A retrospective review of patient demographics and VO2peak data from January 2012 to December 2017 was performed. CAD patients were included if they had successfully completed the supervised 6-month CR program (>75% of exercise prescription) and two VO2peak assessments (respiratory exchange ratio (RER) >1.0). Among all patients, there was an improvement in VO2peak from 21.1 ± 6.3 mL/kg/min to 26.5 ± 7.9 mL/kg/min (+26% ΔVO2peak). Patients in the younger age category (age category 1: 30⁻39 years old) tended to have a greater percent of relative VO2peak improvement when compared to all other age categories (e.g., adults 50 years of age and older). In the regression analysis, VO2peak improvement was associated with younger age (ß = -0.286, p < 0.0001), after adjustment for the baseline VO2peak (ß = -0.456, p < 0.0001), final prescribed exercise speed at CR program completion (ß = 0.254, p < 0.0001), body mass index (ß = -0.172, p < 0.0001), and male sex (ß = 0.153, p < 0.0001). Nonetheless, the study findings indicate that older adults who complete CR may be able to obtain clinically relevant improvements in VO2peak of greater than 20%, and therefore, should be referred for CR.

Antipsychotic discontinuation after the initiation of selective serotonin reuptake inhibitors therapy for the treatment of behavioral and psychological symptoms associated with dementia.

INTRODUCTION: Antipsychotics are used off label to treat behavioral and psychological symptoms of dementia (BPSD). Due to the emerging data of selective serotonin reuptake inhibitors (SSRIs) for treatment of BPSD, clinicians may choose to use this medication class instead of antipsychotics when pharmacologic therapy is necessary. The objective of this study was to evaluate the prevalence of antipsychotic discontinuation 6 months after SSRI initiation for the treatment of BPSD. METHODS: Patients with Alzheimer dementia who were prescribed an antipsychotic and later prescribed an SSRI for BPSD during January 1, 2009, through December 30, 2014, were studied. Exclusion criteria included (1) a dementia diagnosis besides Alzheimer; (2) scheduled benzodiazepines, mood stabilizers, or non-SSRI antidepressant use during the study period; (3) diagnoses of bipolar or psychotic disorders; and (4) diagnosis of delirium during the study period. Patients' age, sex, race, and functional assessment of staging for Alzheimer disease scores were collected. The names, doses, and stop dates of SSRIs and antipsychotics were also recorded. RESULTS: Thirty-six patients were included in the analyses. Overall, antipsychotic use was reduced in 11 patients (30.6%). Ten patients (27.8%) discontinued the antipsychotic, and 1 additional patient had a reduction in dose. When comparing specific SSRIs, 8 (72%) responders were prescribed citalopram, and 15 (60%) nonresponders were prescribed sertraline. DISCUSSION: Approximately 30% of patients with Alzheimer dementia who were prescribed antipsychotics for BPSD were able to discontinue the medication or had a dose reduction after starting SSRI therapy. Most SSRI responders were prescribed citalopram.

Validity of the Center for Epidemiological Studies Depression scale in Type 2 diabetes.

OBJECTIVE: Depressive symptoms are common among people with Type 2 diabetes mellitus (T2DM). This study aimed to validate the 3-factor structure of the 14-item Center for Epidemiological Studies Depression (CES-D) scale proposed by Carleton et al. (2013) in a T2DM population. METHODS: The CES-D was administered to consecutive patients with T2DM entering a rehabilitation program. Construct validity was assessed using confirmatory factor analysis. Subscale viability, differential item functioning, and associations with clinical characteristics were tested in bifactor models. RESULTS: Among adults with T2DM (n=305, age 56.9±11.1, 44.9% male, duration of diabetes 7.8±7.9years, HbA1c 0.076±0.014%), the construct validity of Carleton's 3-factor solution (negative affective, positive affective and somatic symptoms) was confirmed, although negative affective and somatic symptoms were highly correlated (r=0.926). The CES-D items can be summed to arrive at a total score (ωH=0.869), but not subscale scores (ωS>0.7). Differential item functioning was not found based on age or body mass index (BMI), but Item 1 ("I was bothered by things that don't usually bother me") was inflated in women and Item 7 ("I felt that everything I did was an effort") was inflated in those with higher glycosylated haemoglobin (HbA1c). The general depression factor decreased with age (ß=-0.247, p<0.001) and increased with BMI (ß=0.102, p=0.041) but not HbA1c (ß=0.065, p=0.461). Negative affective symptoms (ß=0.743, p=0.001), but not other depressive symptoms, were higher in women. CONCLUSIONS: The 14-item CES-D retained construct validity in adults with T2DM. Depressive symptoms were associated with younger age, female gender and BMI, but not with glycemic control.

Mood and metabolism: Anhedonia as a clinical target in Type 2 diabetes.

Epidemiological evidence suggests a bidirectional relationship between depression and Type 2 diabetes mellitus. In Type 2 diabetes, depression affects behavioural factors such as diet and physical activity that promote positive energy balance and influence diabetes outcomes. Examinations of depressive symptoms by dimension have suggested that anhedonia, the inability to anticipate, seek, choose and enjoy reward, may be of particular clinical importance. Structural and functional brain changes in Type 2 diabetes distributed throughout the principally dopaminergic reward circuitry suggest a neurobiological basis for motivational and decisional aspects of anhedonia. Interrelated neuroendocrine, bio-energetic, oxidative and inflammatory changes suggest mechanisms underlying neuronal damage and dopaminergic deficits. A consequential shift in effort-related reward choices and their effects on energy expenditure, self-care and eating behaviours is suggested to affect Type 2 diabetes outcomes. The clinical implications for screening and psychopharmacology of depressive symptoms in people with Type 2 diabetes are discussed.