Phospho-RNA-Seq Highlights Specific Small RNA Profiles in Plasma Extracellular Vesicles.

Small RNAs (sRNAs) are bioactive molecules that can be detected in biofluids, reflecting physiological and pathological states. In plasma, sRNAs are found within extracellular vesicles (EVs) and in extravesicular compartments, offering potential sources of highly sensitive biomarkers. Deep sequencing strategies to profile sRNAs favor the detection of microRNAs (miRNAs), the best-known class of sRNAs. Phospho-RNA-seq, through the enzymatic treatment of sRNAs with T4 polynucleotide kinase (T4-PNK), has been recently developed to increase the detection of thousands of previously inaccessible RNAs. In this study, we investigated the value of phospho-RNA-seq on both the EVs and extravesicular plasma subfractions. Phospho-RNA-seq increased the proportion of sRNAs used for alignment and highlighted the diversity of the sRNA transcriptome. Unsupervised clustering analysis using sRNA counts matrices correctly classified the EVs and extravesicular samples only in the T4-PNK treated samples, indicating that phospho-RNA-seq stresses the features of sRNAs in each plasma subfraction. Furthermore, T4-PNK treatment emphasized specific miRNA variants differing in the 5'-end (5'-isomiRs) and certain types of tRNA fragments in each plasma fraction. Phospho-RNA-seq increased the number of tissue-specific messenger RNA (mRNA) fragments in the EVs compared with the extravesicular fraction, suggesting that phospho-RNA-seq favors the discovery of tissue-specific sRNAs in EVs. Overall, the present data emphasizes the value of phospho-RNA-seq in uncovering RNA-based biomarkers in EVs.

Relative expression of receptors in uterine natural killer cells compared to peripheral blood natural killer cells.

One would expect maternal immune cells to attack the invading trophoblast as the placenta is semi-allogenic. However, they appear to cooperate with the trophoblast in disrupting the arterial wall which has been determined in several studies. uNK cells are a particular type of immune cell that appears to play a role in pregnancy. As in pregnancy, the key contributors to trophoblast invasion appear to be a unique combination of genes, which appear to regulate multiple components of the interactions between placental and maternal cells, called HLA class 1b genes. The HLA class 1b genes have few alleles, which makes them unlikely to be recognized as foreign by the maternal cells. The low polymorphic properties of these particular HLAs may aid trophoblasts in actively avoiding immune attacks. This review gives a complete description of the mechanisms of interaction between HLAs and maternal uNK cells in humans.

Very low calorie ketogenic diet and common rheumatic disorders: A case report.

BACKGROUND: The scientific literature provides evidence that nutritional ketosis can be an important support in the treatment of pathologies in which inflammation is present, as recent studies have shown that ketone bodies have anti-inflammatory activity in numerous diseases, including rheumatic diseases. We report the case of a 22-year-old woman with class I obesity and juvenile idiopathic arthritis who started treatment with a very low calorie ketogenic diet (VLCKD). CASE SUMMARY: The patient was a 22-year-old woman diagnosed with juvenile idiopathic arthritis at age 4 years and with a body mass index (BMI) of 30.8 kg/m2, waist circumference (WC) 80 cm, fat mass (FM) 28.1 kg, free FM 45.7 kg, and visceral adipose tissue (VAT) 3.5 kg, assessed on bioimpedance analysis. She was treated using a commercial VLCKD weight-loss program (PNK® method); this program provides high-biological-value protein preparations and natural foods. Each protein preparation contains 15 g protein, 4 g carbohydrate, 3 g fat, and 50 mg omega-3 docosahexaenoic acid, with an energy content of 90-120 kcal. After four months on the program, the BMI was 28.6 kg/m2, WC 73 cm, FM 23.2 kg, free FM 41.9 kg, and VAT 2.9 kg. CONCLUSION: VLCKD enabled the patient to reach her target weight and to reduce her joint pain and headaches. Laboratory inflammatory indices also normalized.

Immunomodulatory effect of a very-low-calorie ketogenic diet compared with bariatric surgery and a low-calorie diet in patients with excessive body weight.

BACKGROUND & AIM: Inflammation and oxidative stress are the most probable mechanistic link between obesity and its co-diseases with cancer among them. The aim of this study was to evaluate whether the nutritional ketosis and weight loss induced by a very-low-calorie ketogenic diet (VLCKD) modulates the inflammatory and oxidative stress profile, compared with a standard, balanced hypocaloric diet (LCD) or bariatric surgery (BS) in patients with obesity. METHODS: The study was performed in 79 patients with overweight or obesity and 32 normal-weight volunteers as the control group. Patients with obesity underwent a weight reduction therapy based on VLCKD, LCD or BS. The quantification of the circulating levels of a multiplexing test of cytokines and carcinogenesis/aging biomarkers, as well as of lipid peroxides and total antioxidant power, was carried out. RESULTS: First, we observed that pro-inflammatory cytokines increase, while anti-inflammatory cytokines decrease under excessive body weight. Relevantly, when patients underwent weight loss strategies, it was shown that energy-restricted and surgical strategies of weight loss induced changes in circulating cytokine and lipid peroxides. This effect was more notable in patients following the VLCKD than the LCD or BS and it was observed mainly in the ketosis phase of the intervention. Particularly, IL-11, IL-12, IL-2, INF-γ, INF-ß, Pentraxin-3 or MMP1 changed after VLCKD. Whereas, APRIL, TWEAK, osteocalcin and IL-28A increased after BS. CONCLUSION: As far as we know, this is the first study that evaluate the time-course of cytokines and oxidative stress markers after a VLCKD as compared with a standard LCD and BS. The observed results support the immunomodulatory effect of nutritional ketosis induced by a VLCKD synergistically with weight loss as a strategy to improve innate-immunity and to prevent infections and carcinogenesis in patients with obesity.

CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples.

SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.

Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell-Trophoblast Interactions.

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFß and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFß, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFß, their cytotoxicity increased. In the case of adding TGFß to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFß in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

Effectiveness and Safety of a Very Low-Calorie Ketogenic Diet on Weight Regain Following Bariatric Surgery.

PURPOSE: Obesity is one of the major public health problems worldwide and bariatric surgery is considered the gold standard treatment for severe obesity. Although the literature supports the safety and efficacy of the very low-calorie ketogenic diet (VLCKD) in patients with obesity, there is no study analyzing the use in case of failure or weight regain after bariatric surgery. This work has the purpose of assessing the response to a VLCKD (Pnk® method) in this population. METHODS: We have conducted an observational, retrospective, and descriptive study with the analysis of medical charts of private offices where patients were examined between 2014 and 2019, totalizing 11 participants who underwent Roux-en-Y gastric bypass (RYGB) without a reduction of 50% in body weight excess or any weight regain after the surgery. The patients followed the first stage of a commercial weight loss program (Pnk® method) which consists of a VLCKD (600-800 kcal/day), low in carbohydrates and lipids. RESULTS: A significant reduction was observed (p-value < 0.05) in weight, body mass index (BMI), abdominal circumference (AC), and glycated hemoglobin (A1C) post-ketogenic diet. Uric acid, transaminases, urea, and creatinine values did not show differences between pre- and post-ketosis. During the course of the study, no serious adverse events were reported. CONCLUSION: This study has shown that the ketogenic diet can be recommended as an effective and safe treatment for patients who progressed with insufficient weight loss or regain after bariatric surgery.

Direct oligonucleotide sequencing with nanopores.

Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5' phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5' end of the oligos and to leave a 3' adenosine overhang after binding to their target. The 3' adenosine overhang of the adapter and the terminal phosphate makes the 5' end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.

Efficacy of a 2-Month Very Low-Calorie Ketogenic Diet (VLCKD) Compared to a Standard Low-Calorie Diet in Reducing Visceral and Liver Fat Accumulation in Patients With Obesity.

Background: Currently the treatment of non-alcoholic fatty liver disease (NAFLD) is based on weight loss through lifestyle changes, such as exercise combined with calorie-restricted dieting. Objectives: To assess the effects of a commercially available weight loss program based on a very low-calorie ketogenic diet (VLCKD) on visceral adipose tissue (VAT) and liver fat content compared to a standard low-calorie (LC) diet. As a secondary aim, we evaluated the effect on liver stiffness measurements. Methods: Open, randomized controlled, prospective pilot study. Patients were randomized and treated either with an LC or a VLCKD and received orientation and encouragement to physical activity equally for both groups. VAT, liver fat fraction, and liver stiffness were measured at baseline and after 2 months of treatment using magnetic resonance imaging. Paired t-tests were used for comparison of continuous variables between visits and unpaired test between groups. Categorical variables were compared using the χ2-test. Pearson correlation was used to assess the association between VAT, anthropometric measures, and hepatic fat fraction. A significance level of the results was established at p < 0.05. Results: Thirty-nine patients (20 with VLCKD and 19 with LC) were evaluated at baseline and 2 months of intervention. Relative weight loss at 2 months was -9.59 ± 2.87% in the VLCKD group and -1.87 ± 2.4% in the LC group (p < 0.001). Mean reductions in VAT were -32.0 cm2 for VLCKD group and -12.58 cm2 for LC group (p < 0.05). Reductions in liver fat fraction were significantly more pronounced in the VLCKD group than in the LC group (4.77 vs. 0.79%; p < 0.005). Conclusion: Patients undergoing a VLCKD achieved superior weight loss, with significant VAT and liver fat fraction reductions when compared to the standard LC diet. The weight loss and rapid mobilization of liver fat demonstrated with VLCKD could serve as an effective alternative for the treatment of NAFLD. Clinical Trial Registration: ClinicalTrials.gov, identifier: NCT04322110.

[Multidisciplinary methodology and ketogenic diet in real clinical practice: efficacy and rapidity in weight loss. Survival Analysis PROMET Lipoinflammation study]. / Metodología multidisciplinar y dieta cetogénica en la práctica clínica real: eficacia y rapidez en la pérdida de peso. Análisis de supervivencia. Estudio PROMET Lipoinflamación.

INTRODUCTION: Objective: the aim of the current work was to evaluate the response time to a method of weight loss that includes dietary guidelines, physical exercise and emotional support. The response was defined as a loss of 10% of the baseline weight. Methods: data was obtained from the patients' record recruited in Promet Lipoinflamación, an observational study of real world data in obese or overweight patients treated with a multidisciplinary method and based initially on a very-low-calorie ketogenic (VLCK) diet. Weight loss rate was evaluated through a survival analysis Kaplan-Meier and related factors through Cox regression). Results: 6,369 subjects were included and 74.4% managed to reach a weight loss of 10% in a mean time of 57.64 days (IC 95%: 56.95-58.33). The factors associated with a greater probability of reaching a loss of 10% or more were male gender (RR: 1.37, p < 0.001), obesity types I, II and III vs. overweight (RR: 1.24, p < 0.001, 1.26, p < 0.001 and 1.22, p < 0.001, respectively) and young age vs. more than 55 years old (RR: 2.17, p < 0.001). Conclusion: Results obtained through real clinical practice show that the method produces fast and intense weight loss. Three out of four patients lost at least 10% of body weight in an average of 58 days.

INTRODUCCIÓN: Objetivo: el objetivo del estudio fue evaluar el tiempo de respuesta, definido como pérdida del 10% del peso, al tratamiento con un método multidisciplinar de pérdida de peso que incluye dieta (inicialmente cetogénica), ejercicio físico y soporte emocional. Métodos: los datos se obtuvieron a partir del registro de pacientes reclutados en el estudio Promet Lipoinflamación, un estudio observacional de práctica clínica real en pacientes obesos o con sobrepeso tratados con el método multidisciplinar, basado inicialmente en una dieta cetogénica de muy bajas calorías. La velocidad se valoró mediante un análisis de supervivencia Kaplan-Meier y los factores asociados mediante regresión de Cox. Resultados: la muestra estudiada fue de 6369 sujetos. El 74,4% consiguió alcanzar una pérdida de peso del 10% en una media de tiempo de 57,64 días (IC 95%: 56,95-58,33]. Los factores asociados a mayor probabilidad de alcanzar pérdida del 10% o más fueron género masculino (RR: 1,37; p < 0,001), obesidad de tipos I, II y III frente a sobrepeso (RR: 1,24; p < 0,001; 1,26; p < 0,001 y 1,22; p < 0,001, respectivamente) y edad joven frente a mayor de 55 años (RR: 2,17; p < 0,001). Conclusiones: los resultados obtenidos a través de la práctica clínica real muestran que el método produce una pérdida de peso rápida e intensa. Tres de cada cuatro pacientes perdieron, como mínimo, el 10% del peso corporal en una media de 58 días.

A nanoplatform based on metal-organic frameworks and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity and inhibition.

A nanoplatform based on metal-organic frameworks (MOFs) and lambda exonuclease (λ exo) for the fluorimetric determination of T4 polynucleotide kinase (T4 PNK) activity and inhibition is described. Fe-MIL-88 was selected as the nanomaterial because of its significant preferential binding ability to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) and its quenching property. The synthesized Fe-MIL-88 was characterized by transmission electron microscope, scanning electron microscope, and X-ray photoelectron spectroscopy. In the presence of T4 PNK, FAM-labeled dsDNA (FAM-dsDNA) is phosphorylated on its 5'-terminal. λ exo then recognizes and cleaves the phosphorylated strand yielding FAM-labeled ssDNA (FAM-ssDNA). The fluorescence of the produced FAM-ssDNA is quenched due to Fe-MIL-88's absorbing on FAM-ssDNA. On the contrary, in the absence of T4 PNK, the phosphorylation and cleavage processes cannot take place. Therefore, the fluorescence of FAM-dsDNA still remains. The fluorescence intensity is detected at the maximum emission wavelength of 524 nm using the maximum excitation wavelength of 488 nm. The assay of T4 PNK based on the fluorescence quenching of FAM-ssDNA achieves a linear relationship in the range 0.01-5.0 U mL-1 with a detection limit of 0.0089 U mL-1 in buffer. The assay exhibits excellent performance for T4 PNK activity determination in a complex biological matrix. The results also reveal the ability of the assay for T4 PNK inhibitor screening. Graphical abstract Schematic presentation of a nanoplatform based on Fe-MIL-88 and coupled exonuclease reaction for the fluorimetric determination of T4 polynucleotide kinase activity. FAM-ssDNA, FAM-labeled single-stranded DNA; cDNA, complementary DNA; λ exo, lambda exonuclease;T4 PNK, T4 polynucleotide kinase.

Magnetic bead-gold nanoparticle hybrids probe based on optically countable gold nanoparticles with dark-field microscope for T4 polynucleotide kinase activity assay.

T4 polynucleotide kinase (T4 PNK) plays an essential role in DNA phosphorylation during the DNA repair process. Therefore, the sensitive, selective and convenient detection of T4 PNK activity is of great significance. In this work, we proposed a sensitive non-amplification strategy for the sensing of T4 PNK activity via dark field microscope (DFM) based on magnetic bead (MB)-gold nanoparticle (AuNP) hybrids probe, MB-dsDNA-AuNP (MDA). In the presence of T4 PNK, the 5'-OH termini of DNA are phosphorylated and cleaved into oligonucleotides by lambda exonuclease (λexo), resulting in the destruction of the MDA probe and the separation of AuNP from the MB. Through automatic counting of AuNPs from DFM images, T4 PNK activity can be quantitatively measured. This strategy revealed a limit of detection (LOD) as low as 0.0058 U/mL and exhibited a dynamic range from 0.01 to 1 U/mL. The strategy presents an excellent ability to discriminate T4 PNK from the other proteins and enzymes. Notably, this strategy was applied to screen the T4 PNK inhibitors and test the activity in cell lysates, showing great potential for the discovery of new anticancer drugs and other related research field.

Effect of a Very-Low-Calorie Ketogenic Diet on Circulating Myokine Levels Compared with the Effect of Bariatric Surgery or a Low-Calorie Diet in Patients with Obesity.

: The preservation of muscle mass and muscle function after weight loss therapy is currently a considerable challenge in the fight against obesity. Muscle mass secretes proteins called myokines that have relevant functions in the regulation of metabolism and health. This study was aimed to evaluate whether a very low-calorie ketogenic (VLCK) diet may modulate myokine levels, in addition to changes in body composition, compared to a standard, balanced low-calorie (LC) diet or bariatric surgery in patients with obesity. Body composition, ketosis, insulin sensitivity and myokines were evaluated in 79 patients with overweight/obesity after a therapy to lose weight with a VLCK diet, a LC diet or bariatric surgery. The follow-up was 6 months. The weight loss therapies induced changes in myokine levels in association with changes in body composition and biochemical parameters. The effects on circulating myokine levels compared to those at baseline were stronger after the VLCK diet than LC diet or bariatric surgery. Differences reached statistical significance for IL-8, MMP2 and irisin. In conclusion, nutritional interventions or bariatric surgery to lose weight induces changes in circulating myokine levels, being this effect potentially most notable after following a VLCK diet.

Effect of Synbiotic Supplementation in a Very-Low-Calorie Ketogenic Diet on Weight Loss Achievement and Gut Microbiota: A Randomized Controlled Pilot Study.

SCOPE: Little is known about the changes that a very-low-calorie ketogenic diet (VLCKD) produces in gut microbiota or the effect of synbiotics during the diet. The aim of this study is to evaluate changes in gut microbiota produced by a VLCKD and synbiotic supplementation. METHODS AND RESULTS: A randomized, single-blind, parallel-design trial is conducted in 33 obese patients who follow a weight-loss program (PnK-Method) that include a VLCKD followed by a low-calorie diet (LCD). Subjects are randomly allocated to three groups: one supplemented with synbiotics, a second group supplemented with a placebo during the VLCKD and synbiotics during the LCD phase, and a control group given a placebo. Although symbiotic administration do not produce an effect on microbial diversity, an increase in short-chain fatty aciding producing bacteria and anti-inflammatory mediator signals such as Odoribacter and Lachnospira is shown. The administration of Bifidobacterium animalis subsp. lactis and prebiotics fiber during the LCD is significantly associated with the percentage of weight loss and change in glucose, C-reactive protein and lipopolysaccharide-binding protein. CONCLUSIONS: VLCKD produces important changes in gut microbiota. The administration of synbiotics during VLCKD can improve weight loss through the amelioration of inflammation, which may be mediated by the gut microbiota.

A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe.

The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5' hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650 nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5 U·mL-1), a low detection limit (0.01 U·mL-1) and short assay time (typically 60 min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics. Graphical abstract A turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK) has been developed. It is based on the use of DNA-templated silver nanoclusters (AgNCs). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.

A potential role for peripheral natural killer cell activity induced by preoperative chemotherapy in breast cancer patients.

Tumor-infiltrating lymphocytes are an important prognostic factor after neoadjuvant chemotherapy (NAC) in patients with breast cancer. Natural killer (NK) cells play critical roles in antitumor immune surveillance. Here, we assessed the relationship between peripheral natural killer (pNK) cell activity, tumor microenvironmental factors (TMEFs), and the therapeutic efficacy of preoperative chemotherapy in patients with breast cancer. In a cohort of 39 patients diagnosed with stage II-IV breast cancer who received NAC, we measured pNK cell activity by chromium release assay and assessed TMEF levels by next-generation sequencing. Following NAC, pNK cell activity was increased in 24/39 patients but decreased in 15/39 patients. Increased pNK cell activity following preoperative chemotherapy was associated significantly with the disappearance of axillary lymph node metastasis (Ax+; p = 0.0235). Increased pNK cell activity remained significantly associated with the disappearance of Ax+ in multivariate logistic regression analysis (OR 5.41, 95% CI 1.19-24.52, p = 0.0283). A Grade 2 or higher effect of NAC was associated with high pre-NAC cytotoxic T lymphocyte-associated protein 4 (CTLA-4) levels (p = 0.0281) and elevated post-NAC NK (p = 0.0005) cells and transforming growth factor-beta (TGF-ß; p = 0.0350) levels. The disappearance of Ax+ was associated with high pre-NAC CTLA-4 levels (p = 0.0278) and elevated CD4 levels after NAC (p = 0.0250). The systemic activation of pNK cells after NAC may improve metastatic tumor elimination in patients with breast cancer owing to a release from local immunosuppression, and immune activation in the tumor microenvironment.

Effect of A Very Low-Calorie Ketogenic Diet on Food and Alcohol Cravings, Physical and Sexual Activity, Sleep Disturbances, and Quality of Life in Obese Patients.

Psychological well-being and hunger and food control are two relevant factors involved in the success of weight-loss therapy in treating obesity. Thus, this study aims to evaluate food and alcohol cravings, physical and sexual activity, sleep, and life quality (QoL) in obese patients following a very low-calorie ketogenic (VLCK) diet, as well as the role of weight lost and ketosis on these parameters. A battery of psychological test was performed in twenty obese patients (12 females, 47.2 ± 10.2 year and BMI of 35.5 ± 4.4) through the course of a 4-month VLCK diet on four subsequent visits: baseline, maximum ketosis, reduced ketosis, and endpoint. Each subject acted as their own control. Relevantly, the dietary-induced changes in body composition (7.7 units of BMI lost, 18 kg of fat mass (1.2 kg of visceral fat mass)) were associated with a statistically significant improvement in food craving scores, physical activity, sleepiness, and female sexual function. Overall, these results also translated in a notable enhancement in QoL of the treated obese patients. Therefore, the rapid and sustained weight and fat mass (FM) loss induced by the VLCK diet is associated with good food control and improvements in the psychological well-being parameters in obese subjects, which could contribute to the long-term success of this therapy.

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform.

As a vital enzyme in DNA phosphorylation and restoration, T4 polynucleotide kinase (T4 PNK) has aroused great interest in recent years. Therefore, numerous strategies have been established for highly sensitive detection of T4 PNK based on diverse signal amplification techniques. However, they often need sophisticated design, a variety of auxiliary reagents and enzymes, or cumbersome manipulations. We have designed a new kind of allosteric aptamer probe (AAP) consisting of streptavidin (SA) aptamer and the complementary DNA (cDNA) for simple detection of T4 PNK without signal amplification and with minimized interference in complex biological samples. When the 5'-terminus of the cDNA is phosphorylated by T4 PNK, the cDNA is degraded by lambda exonuclease to release the fluorescein amidite (FAM)-labeled SA aptamer, which subsequently binds to streptavidin beads. The enhancement of the fluorescence signal on SA beads can be detected precisely and easily by a microscope or flow cytometer. Our method performs well in complex biological samples as a result of the enrichment of the signaling molecules on beads, as well as simple manipulations to discard the background interference and nonbinding molecules. Without signal amplification techniques, our AAP method not only avoids complicated manipulations but also decreases the time required. With the advantages of ease of operation, reliability, and robustness for T4 PNK detection in buffer as well as real biological samples, the AAP has great potential for clinical diagnostics, inhibitor screening, and drug discovery.

Axl signaling induces development of natural killer cells in vitro and in vivo.

Natural killer (NK) cells have been well known to play a critical role in innate immunity, but they are also capable of regulating adaptive immunity through the induction of T cell-mediated memory response and B cell-mediated autoimmune response. NK cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow (BM), and a series of surface molecules are expressed on NK cells in a differentiation stage-specific manner. Axl receptor tyrosine kinase is originally identified as homeostatic regulators for antigen-presenting cells, and its ligand, growth-arrest-specific gene 6 (Gas6), has been reported to promote cell survival, proliferation, and migration, but their regulatory role in the development and effector function of NK cells is not yet fully understood. In this study, to investigate whether Axl is required for the regulation of NK cell development, the expression of mature NK (mNK) cell-specific receptors and NK cell-associated genes was analyzed in the differentiated HSCs-derived NK cells in vitro and the NK cells harvested from Axl-/- mice. We found that agonistic anti-Axl antibody or recombinant Gas6 specifically upregulated the expression of mNK cell-specific receptors, such as LY49A, Ly49G2, Ly49C/F/I, NKG2A/C/E (1.5- to 3.5-fold increase), and NK cell-associated genes, such as IL-2Rß (2.3- or 2.4-fold increase), Perforin (4.1- or 2.1-fold increase), IL-15Rα (2.14- or 2.04-fold increase), and IFN-γ (3.3- or 2.8-fold increase) compared to each isotype control, whereas it was abrogated by treatment of Axl-Ig. Anti-Axl antibody or rGas6 also induced a 2.5- or 1.9-fold increase in the proliferation of developing NK cells compared to each control, respectively. mNK cell populations expressing mNK cell-specific receptors were reduced about twofold in NK cells differentiated from HSCs of Axl-/- mice compared with those of wild-type mice. Furthermore, the triggering of Axl signaling by agonistic anti-Axl antibody promoted the cytolytic activity (1.5- to 1.9-fold increase) against target tumor cells. In B16F10 melanoma-bearing mice, the number of metastatic colonies was decreased by 83 % by the administration of mNK cells treated with anti-Axl antibody compared to control Ig. These data suggest that Axl plays an essential role in the regulation of NK cell development as well as NK effector function.

Effect of DHA supplementation in a very low-calorie ketogenic diet in the treatment of obesity: a randomized clinical trial.

A VLCK diet supplemented with DHA, commercially available, was tested against an isocaloric VLCK diet without DHA. The main purpose of this study was to compare the effect of DHA supplementation in classic cardiovascular risk factors, adipokine levels, and inflammation-resolving eicosanoids. A total of obese patients were randomized into two groups: a group supplemented with DHA (n = 14) (PnK-DHA group) versus a group with an isocaloric diet free of supplementation (n = 15) (control group). The follow-up period was 6 months. The average weight loss after 6 months of treatment was 20.36 ± 5.02 kg in control group and 19.74 ± 5.10 kg in PnK-DHA group, without statistical differences between both groups. The VLCK diets induced a significant change in some of the biological parameters, such as insulin, HOMA-IR, triglycerides, LDL cholesterol, C-reactive protein, resistin, TNF alpha, and leptin. Following DHA supplementation, the DHA-derived oxylipins were significantly increased in the intervention group. The ratio of proresolution/proinflammatory lipid markers was increased in plasma of the intervention group over the entire study. Similarly, the mean ratios of AA/EPA and AA/DHA in erythrocyte membranes were dramatically reduced in the PnK-DHA group and the anti-inflammatory fatty acid index (AIFAI) was consistently increased after the DHA treatment (p < 0.05). The present study demonstrated that a very low-calorie ketogenic diet supplemented with DHA was significantly superior in the anti-inflammatory effect, without statistical differences in weight loss and metabolic improvement.