Interspecies Chimerism with Mammalian Pluripotent Stem Cells.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

In Vivo Amelioration of Age-Associated Hallmarks by Partial Reprogramming.

Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.

Novel synthetic polymyxins kill Gram-positive bacteria.

Background: Staphylococcus aureus, including 'superbug' MRSA, is a major cause of nosocomial infections. In the European Union, up to 171 200 new nosocomial MRSA infections are acquired annually, and in the USA S. aureus causes more deaths than HIV/AIDS and tuberculosis combined. MRSA is also the first group of pathogens that infect the pulmonary tract in young patients with cystic fibrosis. Objectives: We describe two newly developed and synthesized colistin (polymyxin E)-inspired molecules. Methods: A collection of several isolates of S. aureus [including MRSA and vancomycin-resistant S. aureus (VRSA)] was tested. To check the antimicrobial activity, we performed time-kill curves, growth curves, biofilm eradication, toxicity and isothermal titration calorimetry. Results: Both peptides showed high antimicrobial activities (MIC 4 mg/L) and low relative toxicities (selectivity index close to 23). Conclusions: Successful production of polymyxin-scaffold molecules active against S. aureus, both MRSA and VRSA, opens up new approaches to the treatment of these complicated infections.

Comparative study of the oral absorption of microencapsulated ferric saccharate and ferrous sulfate in humans.

PURPOSE: Our objective was to compare the absorption of microencapsulated ferric saccharate (MFS) and ferrous sulfate (FS) in a fortified milk product, using a crossover design. METHODS: Seventeen non-iron-deficient healthy adults from both sexes participated in the study. On each intervention day (days 1 and 8), after an overnight fast, the volunteers consumed one type of product (test or control) and blood sampling was carried out at different times. The interventions days were separated by 7-day washout periods. This study was double blinded, crossover and randomized for nature of the test meals. The primary outcomes of the study were total serum iron and transferrin saturation. RESULTS: No significant differences could be observed in serum iron concentration during the 6-h postprandial study due to the type of milk product consumed, and there was neither an effect of time nor an interaction between the type of milk product and time. Transferrin saturation significantly increased after the intake of both products (P < 0.005), reaching a peak value between hours 2 and 4. No significant differences were detected between MFS and FS, indicating that iron absorption from MFS is equivalent to absorption from FS. CONCLUSIONS: MFS is a new ingredient that allows the fortification of a wide range of food products, including heat-processed and non-acidic products with similar absorption to FS, designed to produce neither organoleptic changes nor off-color development during storage of fortified food.

Intervention with metabolites emulating endogenous cell transitions accelerates muscle regeneration in young and aged mice.

Tissue regeneration following an injury requires dynamic cell-state transitions that allow for establishing the cell identities required for the restoration of tissue homeostasis and function. Here, we present a biochemical intervention that induces an intermediate cell state mirroring a transition identified during normal differentiation of myoblasts and other multipotent and pluripotent cells to mature cells. When applied in somatic differentiated cells, the intervention, composed of one-carbon metabolites, reduces some dedifferentiation markers without losing the lineage identity, thus inducing limited reprogramming into a more flexible cell state. Moreover, the intervention enabled accelerated repair after muscle injury in young and aged mice. Overall, our study uncovers a conserved biochemical transitional phase that enhances cellular plasticity in vivo and hints at potential and scalable biochemical interventions of use in regenerative medicine and rejuvenation interventions that may be more tractable than genetic ones.

Molecular identity of the lateral lemniscus nuclei in the adult mouse brain.

The dorsal (DLL), intermediate (ILL), and ventral (VLL) lateral lemniscus nuclei are relay centers in the central auditory pathway of the brainstem, commonly referred to as the lateral lemniscus nuclei (LLN). The LLN are situated in the prepontine and pontine hindbrain, from rhombomeres 1 to 4, extending from the more rostral DLL to the caudal VLL, with the ILL lying in between. These nuclei can be distinguished morphologically and by topological and connectivity criteria, and here, we set out to further characterize the molecular nature of each LLN. We searched in situ hybridization studies in the Allen Mouse Brain Atlas for genes differentially expressed along the rostrocaudal axis of the brainstem, identifying 36 genes from diverse functional families expressed in the LLN. Available information in the databases indicated that 7 of these 36 genes are either associated with or potentially related to hearing disorders. In conclusion, the LLN are characterized by specific molecular profiles that reflect their rostrocaudal organization into the three constituent nuclei. This molecular regionalization may be involved in the etiology of some hearing disorders, in accordance with previous functional studies of these genes.

Studying Lipid Membrane Interactions of a Super-Cationic Peptide in Model Membranes and Living Bacteria.

The super-cationic peptide dendrimers (SCPD) family is a valuable class of antimicrobial peptide candidates for the future development of antibacterial agents against multidrug-resistant gram-negative bacteria. The deep knowledge of their mechanism of action is a major challenge in research, since it may be the basis for future modifications/optimizations. In this work we have explored the interaction between SCPD and membranes through biophysical and microbiological approaches in the case of the G1OLO-L2OL2 peptide. Results support the idea that the peptide is not only adsorbed or close to the surface of the membrane but associated/absorbed to some extent to the hydrophobic-hydrophilic region of the phospholipids. The presence of low concentrations of the peptide at the surface level is concomitant with destabilization of the cell integrity and this may contribute to osmotic stress, although other mechanisms of action cannot be ruled out.

In vivo partial reprogramming alters age-associated molecular changes during physiological aging in mice.

Partial reprogramming by expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) for short periods of time restores a youthful epigenetic signature to aging cells and extends the life span of a premature aging mouse model. However, the effects of longer-term partial reprogramming in physiologically aging wild-type mice are unknown. Here, we performed various long-term partial reprogramming regimens, including different onset timings, during physiological aging. Long-term partial reprogramming lead to rejuvenating effects in different tissues, such as the kidney and skin, and at the organismal level; duration of the treatment determined the extent of the beneficial effects. The rejuvenating effects were associated with a reversion of the epigenetic clock and metabolic and transcriptomic changes, including reduced expression of genes involved in the inflammation, senescence and stress response pathways. Overall, our observations indicate that partial reprogramming protocols can be designed to be safe and effective in preventing age-related physiological changes. We further conclude that longer-term partial reprogramming regimens are more effective in delaying aging phenotypes than short-term reprogramming.

In vivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Molecular Segmentation of the Spinal Trigeminal Nucleus in the Adult Mouse Brain.

The trigeminal column is a hindbrain structure formed by second order sensory neurons that receive afferences from trigeminal primary (ganglionic) nerve fibers. Classical studies subdivide it into the principal sensory trigeminal nucleus located next to the pontine nerve root, and the spinal trigeminal nucleus which in turn consists of oral, interpolar and caudal subnuclei. On the other hand, according to the prosomeric model, this column would be subdivided into segmental units derived from respective rhombomeres. Experimental studies have mapped the principal sensory trigeminal nucleus to pontine rhombomeres (r) r2-r3 in the mouse. The spinal trigeminal nucleus emerges as a plurisegmental formation covering several rhombomeres (r4 to r11 in mice) across pontine, retropontine and medullary hindbrain regions. In the present work we reexamined the issue of rhombomeric vs. classical subdivisions of this column. To this end, we analyzed its subdivisions in an AZIN2-lacZ transgenic mouse, known as a reference model for hindbrain topography, together with transgenic reporter lines for trigeminal fibers. We screened as well for genes differentially expressed along the axial dimension of this structure in the adult and juvenile mouse brain. This analysis yielded genes from multiple functional families that display transverse domains fitting the mentioned rhombomeric map. The spinal trigeminal nucleus thus represents a plurisegmental structure with a series of distinct neuromeric units having unique combinatorial molecular profiles.

Multiple Regionalized Genes and Their Putative Networks in the Interpeduncular Nucleus Suggest Complex Mechanisms of Neuron Development and Axon Guidance.

The interpeduncular nucleus (IPN) is a highly conserved limbic structure in the vertebrate brain, located in the isthmus and rhombomere 1. It is formed by various populations that migrate from different sites to the distinct domains within the IPN: the prodromal, rostral interpeduncular, and caudal interpeduncular nuclei. The aim here was to identify genes that are differentially expressed across these domains, characterizing their putative functional roles and interactions. To this end, we screened the 2,038 genes in the Allen Developing Mouse Brain Atlas database expressed at E18.5 and we identified 135 genes expressed within the IPN. The functional analysis of these genes highlighted an overrepresentation of gene families related to neuron development, cell morphogenesis and axon guidance. The interactome analysis within each IPN domain yielded specific networks that mainly involve members of the ephrin/Eph and Cadherin families, transcription factors and molecules related to synaptic neurotransmission. These results bring to light specific mechanisms that might participate in the formation, molecular regionalization, axon guidance and connectivity of the different IPN domains. This genoarchitectonic model of the IPN enables data on gene expression and interactions to be integrated and interpreted, providing a basis for the further study of the connectivity and function of this poorly understood nuclear complex under both normal and pathological conditions.

Cranioplasty with Autologous Bone Flaps Cryopreserved with Dimethylsulphoxide: Does Tissue Processing Matter.

OBJECTIVE: The aim of this article was to study the outcome of patients who underwent cranioplasty with cryopreserved autologous bone after decompressive craniectomy. METHODS: Data from 74 patients were retrospectively analyzed. They were divided into groups according to the storage time and the age at cranioplasty. To assess the predictive potential for complication, factors were related to successive stages (preoperative, craniectomy, tissue processing, cranioplasty, and postoperative). Cooling and warming rates applied on bone flap were calculated. The ability to inhibit microbial growth was determined exposing bone fragments to a panel of microorganisms. The concentration of antibiotics eluted from the bone was also determined. A bone explant culture method was used to detect living cells in the thawed cranial bone. RESULTS: Hydrocephalus was significantly more frequent in pediatric patients (26.7%) than in adults (5.1%). The overall rate of bone flap resorption was 21.6% (43.7% of which required reoperation). Surgical site infection after cranioplasty was detected in 6.8% of patients. There was no correlation between infection as a postoperative complication and previous microbiological-positive culture during processing. The cause of craniectomy did not influence the risk of bone flap contamination. Vancomycin was the only antibiotic detected in the supernatant where the bone was incubated. Outgrowth from bone explants was observed in 36.8% of thawed skulls. An early start of bone flap processing at the tissue bank had a positive effect on cell viability. CONCLUSIONS: The outcome after autologous cranioplasty is a multifactorial process, which is modulated by patient-related, surgery-related, and bone-related factors.

Super-Cationic Peptide Dendrimers-Synthesis and Evaluation as Antimicrobial Agents.

Microbial infections are a major public health concern. Antimicrobial peptides (AMPs) have been demonstrated to be a plausible alternative to the current arsenal of drugs that has become inefficient due to multidrug resistance. Herein we describe a new AMP family, namely the super-cationic peptide dendrimers (SCPDs). Although all members of the series exert some antibacterial activity, we propose that special attention should be given to (KLK)2KLLKLL-NH2 (G1KLK-L2KL2), which shows selectivity for Gram-negative bacteria and virtually no cytotoxicity in HepG2 and HEK293. These results reinforce the validity of the SCPD family as a valuable class of AMP and support G1KLK-L2KL2 as a strong lead candidate for the future development of an antibacterial agent against Gram-negative bacteria.

In vivo partial reprogramming of myofibers promotes muscle regeneration by remodeling the stem cell niche.

Short-term, systemic expression of the Yamanaka reprogramming factors (Oct-3/4, Sox2, Klf4 and c-Myc [OSKM]) has been shown to rejuvenate aging cells and promote tissue regeneration in vivo. However, the mechanisms by which OSKM promotes tissue regeneration are unknown. In this work, we focus on a specific tissue and demonstrate that local expression of OSKM, specifically in myofibers, induces the activation of muscle stem cells or satellite cells (SCs), which accelerates muscle regeneration in young mice. In contrast, expressing OSKM directly in SCs does not improve muscle regeneration. Mechanistically, expressing OSKM in myofibers regulates the expression of genes important for the SC microenvironment, including upregulation of p21, which in turn downregulates Wnt4. This is critical because Wnt4 is secreted by myofibers to maintain SC quiescence. Thus, short-term induction of the Yamanaka factors in myofibers may promote tissue regeneration by modifying the stem cell niche.

Patient's pre-operative dental anxiety is related to diastolic blood pressure and the need for post-surgical analgesia.

In order to study the relationship of the patient's anxiety level from Corah's Dental Anxiety Scale (DAS) vs different physiological parameters: pre and post-operative blood pressure, and pre and post-operative heart rates, and subsequently, relate the results to the patient's post-operative anti-inflammatory analgesic need, 185 patients requiring a simple dental extraction were recruited. They filled out the DAS in the waiting room prior to their procedure and once in the examination room, their preoperative blood pressure and heart rate was measured. Once the dental extraction had been completed, their blood pressure and heart rate were measured again. Before leaving the clinic, the patient was given an analgesic form in which they had to indicate whether or not they had required analgesia after the procedure. Diastolic blood pressure (DBP) showed statistically significant differences between pre-operative and post-operative (P = 0.001). DAS was related with pre-operative diastolic blood pressure (pre-DBP) (P = 0.001) and post-operative diastolic blood pressure (post-DBP) as well as pre-operative heart rate (pre-HR) (P = 0.027) and post-operative heart rate (post-HR) (P = 0.013). Patients with high levels of DAS tend to take more Ibuprofen 400 mg (P = 0.038). The different levels of anxiety will determine what type of anti-inflammatory analgesia the patient will take, if necessary.

Lactate in the Tumor Microenvironment: An Essential Molecule in Cancer Progression and Treatment.

Cancer is a complex disease that includes the reprogramming of metabolic pathways by malignant proliferating cells, including those affecting the tumor microenvironment (TME). The "TME concept" was introduced in recognition of the roles played by factors other than tumor cells in cancer progression. In response to the hypoxic or semi-hypoxic characteristic of the TME, cancer cells generate a large amount of lactate via the metabolism of glucose and glutamine. Export of this newly generated lactate by the tumor cells together with H+ prevents intracellular acidification but acidifies the TME. In recent years, the importance of lactate and acidosis in carcinogenesis has gained increasing attention, including the role of lactate as a tumor-promoting metabolite. Here we review the existing literature on lactate metabolism in tumor cells and the ability of extracellular lactate to direct the metabolic reprogramming of those cells. Studies demonstrating the roles of lactate in biological processes that drive or sustain carcinogenesis (tumor promotion, angiogenesis, metastasis and tumor resistance) and lactate's role as an immunosuppressor that contributes to tumor evasion are also considered. Finally, we consider recent therapeutic efforts using available drugs directed at and interfering with lactate production and transport in cancer treatment.

Netrin-1/DCC Signaling Differentially Regulates the Migration of Pax7, Nkx6.1, Irx2, Otp, and Otx2 Cell Populations in the Developing Interpeduncular Nucleus.

The interpeduncular nucleus (IPN) is a hindbrain structure formed by three main subdivisions, the prodromal (Pro) domain located at the isthmus (Ist), and the rostral and caudal interpeduncular domains (IPR, IPC) within rhombomere 1 (r1). Various cell populations can be detected in the IPN through the expression of the Nkx6.1, Otp, Otx2, Pax7, and/or Irx2 transcription factors. These cell populations follow independent dorsoventral tangential and radial migratory routes targeting the ventral paramedian region of Ist and r1. Here we set out to examine the influence of the Netrin-1/DCC pathway on these migrations, since it is known to regulate other processes of neuronal migration in the brain. To this end, we analyzed IPN development in late gestational wild-type and DCC-/- mice, using mainly in situ hybridization (ISH) to identify the cells expressing each of the aforementioned genes. We found that the migration of Nkx6.1 + and Irx2 + cells into the Pro domain was strongly disrupted by the loss of DCC, as occurred with the migration of Pax7 +, Irx2 +, and Otp + cells that would normally form the IPR. In addition, there was mild impairment of the migration of the Pax7 + and Otx2 + cells that form the IPC. These results demonstrate that the Netrin-1/DCC signaling pathway is involved in the migration of most of the IPN populations, mainly affecting those of the Pro and IPR domains of this nucleus. There are psychiatric disorders that involve the medial habenula (mHb)-IPN system, so that this experimental model could provide a basis to study their neurodevelopmental etiology.

In vivo randomized trial of three marketed milk preparations enriched with calcium and vitamins (D and K) on bone mass and bone turnover markers from biological fluids in premenopausal Caucasian women.

INTRODUCTION: osteoporosis is a metabolic bone disease that leads to increased bone fragility and increased risk of fracture. OBJECTIVES: the aim of the present research was to determine the effectiveness of a diary intake of three different dairy products (250 ml) enriched with vitamins and calcium on decreasing bone mass. METHOS: the present study is a comparative trial of three dairy products fortified with calcium and vitamin D, parallel, randomized, double-blind andsingle-center. Bone mass content (BMC), bone mass density (BMD), T-score and Z-score were measured in different locations, besides biochemical markers along 18 months in premenopausal women. Two hundred and ten volunteers from all the three groups were submitted to the same monitoring procedures, consisting on blood extraction, urine collection and energy X-ray absorptiometry (DEXA) done in the laboratory. The monitoring was carried on three times, first at month 0 (baseline), the second at month 9 (in the middle of the treatment) and, finally, at month 18 (the end of the treatment). RESULTS: the majority of anatomical locations showed both BMC and BMD decrease ranging between 0.5% and 1.5%. The T-score and the Z-scoreincreased in lumbar spine after the treatment with the dairy products. Moreover, the most noteworthy change on the biomarkers of bone resorption was showed by plasmatic tartrate-resistant acid phosphatase (TRAP), with and increase between 20.7% and 29.5% after the intake of the different products. CONCLUSIONS: therefore, the intake of the three dairy products improves the bone mass in lumbar spine, leading to important changes in the concentration of biomarkers of bone resorption. Especially, tartrate-resistant acid phosphatase seems to be strongly influenced by the intake of every dairy product. However, no significant differences were found between the different dairy products used in the present study. Therefore, the intake of dairy product seems to be more determinant than micronutrients supplementation.

Electrophoretic techniques applied to the detection and analysis of the human microsatellite DG10s478.

Short nucleotide repetitions (STRs) are commonly used as genetic markers; thus their detection and analysis constitutes a very important tool for the mapping of genetic diseases, as well as for gathering information about genetic polymorphisms at the population level. STRs can be detected with agarose- or acrylamide-based electrophoretic techniques, followed by visualization of the DNA sample with ethidium bromide, silver nitrate, or fluorophore labeling. In this work, we analyzed genomic DNA from five individuals affected with type II diabetes mellitus (T2DM) and five controls (unaffected individuals) in order to know the most precise and reproducible technique for the analysis of the existing polymorphism in the STR DG10S478 of the TCF7L2 gene. The combination of PCR with labeling of the products with the CY5 fluorophore, followed by detection on an ALFexpress sequencer, offered the required resolution to detect the variability in this STR, based solely on size analysis. Our methodology offers similar accuracy and reproducibility at lower costs than existing methods based on the sequencing of PCR products, and is a faster alternative when applied to genotyping studies.

[Effect of quinua (Chenopodium quinoa)consumption as a coadjuvant in nutritional intervention in prediabetic subjects]. / Efecto del consumo de quinua (Chenopodium quinoa)como coadyuvante en la intervención nutricional en sujetos prediabéticos.

INTRODUCTION: Quinoa is a pseudocereal containing low glycemic index carbohydrates, dietary fiber, high biological value protein, phytosterols, and n-3 and n-6 fatty acids, which has generated interest in prediabetes nutritional interventions. This randomized (2:1), placebo-controlled, double-blind study evaluated the effects of processed quinoa on body mass index (BMI), glycated hemoglobin (HbA1c), fasting plasma glucose (FPG) and the satiation and fullness (complete) degree in prediabetic patients. MATERIAL AND METHOD: Thirty patients were randomized (2:1) in two study arms: Kuska Active product (processed quinoa) and placebo (maltodextrin), with an intake period of 28 days. BMI, HbA1c and FPG were determined before starting treatment and at 28-day intake. Satiety and fullness sensation were just assessed by visual analog scale (VAS) at the day 28. ANOVA was performed for repeated measures with two factors to study (within-subject factor: time; intersubject factor: product consumed) to demonstrate the effectiveness of processed quinoa on the study variables. RESULTS: Twenty-nine patients (placebo, n = 10; quinoa, n = 19) completed the study, and the quinoa group shows a significant decrease in BMI (p < 0.05) and HbA1c values (p < 0.001), and an increase in the satiation and fullness (complete) degree (p < 0.001). No significant differences were found in FPG levels from baseline to post-intake period. CONCLUSIONS: The results show that processed quinoa intake during 28 days decreases BMI and HbA1c levels, maintains FPG levels, and incr eases the satiation and fullness (complete) degree in prediabetic patients.