Significance of clinical-immunological patterns and diagnostic yield of biopsies in microscopic polyangiitis and granulomatosis with polyangiitis.

BACKGROUND: Microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) are the two major antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). OBJECTIVES: To characterize a homogenous AAV cohort and to assess the impact of clinicopathological profiles and ANCA serotypes on clinical presentation and prognosis. Clinical differences in GPA patients according to ANCA serotype and the diagnostic yield for vasculitis of biopsies in different territories were also investigated. RESULTS: This retrospective study (2000-2021) included 152 patients with AAV (77 MPA/75 GPA). MPA patients (96.1% myeloperoxidase [MPO]-ANCA and 2.6% proteinase 3 [PR3]-ANCA) presented more often with weight loss, myalgia, renal involvement, interstitial lung disease (ILD), cutaneous purpura, and peripheral nerve involvement. Patients with GPA (44% PR3-ANCA, 33.3% MPO, and 22.7% negative/atypical ANCA) presented more commonly with ear, nose, and throat and eye/orbital manifestations, more relapses, and higher survival than patients with MPA. GPA was the only independent risk factor for relapse. Poor survival predictors were older age at diagnosis and peripheral nerve involvement. ANCA serotypes differentiated clinical features in a lesser degree than clinical phenotypes. A mean of 1.5 biopsies were performed in 93.4% of patients in different territories. Overall, vasculitis was identified in 80.3% (97.3% in MPA and 61.8% in GPA) of patients. CONCLUSIONS: The identification of GPA presentations associated with MPO-ANCA and awareness of risk factors for relapse and mortality are important to guide proper therapeutic strategies in AAV patients. Biopsies of different affected territories should be pursued in difficult-to-diagnose patients based on their significant diagnostic yield.

Anorectal physiology and colonic motility in children with a history of tethered cord syndrome.

OBJECTIVES: The understanding of the impact of tethered cord syndrome (TCS) on the physiology of the colorectal area is limited. Our aim was to describe anorectal and colonic motility in children with TCS and compare the findings to those of children with functional constipation (FC). METHODS: We conducted a retrospective review of children with TCS who had an anorectal manometry (ARM) performed at our institution from January 2011 to September 2023. We recorded demographics, medical and surgical history, clinical symptoms, and treatment at time of ARM, ARM findings (resting pressure, push maneuver, rectal sensation, rectoanal inhibitory reflex [RAIR], and RAIR duration), and the final interpretation of colonic manometry (CM) if performed. We identified age and sex-matched control groups of children with FC. RESULTS: We included 24 children with TCS (50% female) who had ARM testing (median age at ARM 6.0 years, interquartile range 4.0-11.8 years). All children had constipation at time of ARM. Nineteen children had detethering surgery before ARM was performed. No significant differences in ARM parameters were found between children who had detethering surgery before ARM and children with FC. Among the 24 children, 14 also had a CM performed (13/14 after detethering surgery). No significant differences in colonic motility were found between children with a history of TCS and children with FC. CONCLUSIONS: Anorectal physiology and colonic motility are similar between children with a history of TCS and children with FC, suggesting that the underlying pathophysiology of defecatory disorders in children with and without history of TCS is similar.

Current practice in the care of children with functional constipation: What is the hold up?

OBJECTIVES: For children with intractable functional constipation (FC), there are no evidence-based guidelines for subsequent evaluation and treatment. Our objective was to assess the practice patterns of a large, international cohort of pediatric gastroenterologists. METHODS: We administered a survey to physicians who attended the 2nd World Congress of Pediatric Neurogastroenterology and Motility held in Columbus, Ohio (USA) in September 2023. The survey included 29 questions on diagnostic testing, nonpharmacological and pharmacological treatment, and surgical options for children with intractable FC. RESULTS: Ninety physicians from 18 countries completed the survey. For children with intractable FC, anorectal manometry was the most commonly used diagnostic test. North American responders were more likely than Europeans to use stimulant laxatives (97% vs. 77%, p = 0.032), prosecretory medications (69% vs. 8%, p < 0.001), and antegrade continence enemas (ACE; 83% vs. 46%, p = 0.009) for management. Europeans were more likely than North Americans to require colonic transit testing before surgery (85% vs. 30%, p < 0.001). We found major differences in management practices between Americans and the rest of the world, including use of prosecretory drugs (73% vs. 7%, p < 0.001), anal botulinum toxin injections (81% vs. 58%, p = 0.018), ACE (81% vs. 58% p = 0.018), diverting ileostomies (56% vs. 26%, p = 0.006), and colonic resections (42% vs. 16%, p = 0.012). No differences were found when respondents were compared by years of experience. CONCLUSIONS: Practice patterns in the evaluation and treatment of children with intractable FC differ widely among pediatric gastroenterologists from around the world. A clinical guideline regarding diagnostic testing and surgical decision-making is needed.

Ineffective esophageal motility: Characterization and outcomes across pediatric neurogastroenterology and motility centers in the United States.

OBJECTIVES: Ineffective esophageal motility (IEM) on high-resolution manometry (HRM) is not consistently associated with specific clinical syndromes or outcomes. We evaluated the prevalence, clinical features, management, and outcomes of pediatric IEM patients across the United States. METHODS: Clinical and manometric characteristics of children undergoing esophageal HRM during 2021-2022 were collected from 12 pediatric motility centers. Clinical presentation, test results, management strategies, and outcomes were compared between children with IEM and normal HRM. RESULTS: Of 236 children (median age 15 years, 63.6% female, 79.2% Caucasian), 62 (23.6%) patients had IEM, and 174 (73.7%) patients had normal HRM, with similar demographics, medical history, clinical presentation, and median symptom duration. Reflux monitoring was performed more often for IEM patients (25.8% vs. 8.6%, p = 0.002), but other adjunctive testing was similar. Among 101 patients with follow-up, symptomatic cohorts declined in both groups in relation to the initial presentation (p > 0.107 for each comparison) with management targeting symptoms, particularly acid suppression. Though prokinetics were used more often and behavioral therapy less often in IEM (p ≤ 0.015 for each comparison), symptom outcomes were similar between IEM and normal HRM. Despite a higher proportion with residual dysphagia on follow-up in IEM (64.0% vs. 39.1%, p = 0.043), an alternate mechanism for dysphagia was identified more often in IEM (68.8%) compared to normal HRM (27.8%, p = 0.017). CONCLUSIONS: IEM is a descriptive manometric pattern rather than a clinical diagnosis requiring specific intervention in children. Management based on clinical presentation provides consistent symptom outcomes.

Cobalt protoporphyrin modulates antioxidant enzyme activity in the hypothalamus and motor cortex of female rats.

Cobalt protoporphyrin (CoPP) is a synthetic heme analog that has been observed to reduce food intake and promote sustained weight loss. While the precise mechanisms responsible for these effects remain elusive, earlier research has hinted at the potential involvement of nitric oxide synthase in the hypothalamus. This study aimed to delve into CoPP's impact on the activities of crucial antioxidant enzymes: superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) across seven distinct brain regions (hippocampus, hypothalamus, prefrontal cortex, motor cortex, striatum, midbrain, and cerebellum), as well as in the liver and kidneys. Female Wistar rats weighing 180 to 200 grams received a single subcutaneous dose of 25 µmol/kg CoPP. After six days, brain tissue was extracted to assess the activities of antioxidant enzymes and quantify malondialdehyde levels. Our findings confirm that CoPP administration triggers the characteristic effects of decreased food intake and reduced body weight. Moreover, it led to an increase in SOD activity in the hypothalamus, a pivotal brain region associated with food intake regulation. Notably, CoPP-treated rats exhibited elevated enzymatic activity of catalase, GR, and GST in the motor cortex without concurrent signs of heightened oxidative stress. These results underscore a strong connection between the antioxidant system and food intake regulation. They also emphasize the need for further investigation into the roles of antioxidant enzymes in modulating food intake and the ensuing weight loss, using CoPP as a valuable research tool.

Model of micro-metastases of breast cancer cells in ovarian tissue: Cryopreservation of ZR-75-1 and MDA-MB-231 cells with increased speed of warming increases malignancy.

In medicine, ovarian tissue cryopreservation exists for fertility preservation of cancer patients. In fact, ovarian tissue frozen for subsequent thawing and re-transplantation can be contaminated with cancer cells. Therefore, investigations on the effect of cryopreservation on the post-thawed viability of such cells are relevant. Speed of warming is a key parameter of cell cryopreservation. However, the data about comparative viability of cancer cells cryopreserved with different parameters of warming are limited. The aim of our investigations was to assess the malignancy of cryopreserved cancer cells after conventional cooling followed by relatively slow and quick speed of warming. In vitro cultured breast cancer cells of lines ZR-75-1 and MD0MD-231 in form of compacted fragments (as a model of solid tumors) were frozen following a protocol usually used for freezing of ovarian tissue (6 % ethylene glycol+6 % glycerol+0.15 M sucrose, -0.3 °C/min). Cells were warmed by two routine regimes of warming: at 37 °C ("slow" warming) and 100 °C ("quick" warming). Biological properties of cells were investigated: viability, proliferation rate, 2D- and 3D-migration, transmembrane movement and invasion. Quick warming at 100 °C in comparison with slow warming at 37 °C exhibited significantly higher cell survival for MDA-MB-231 cells: 70.1 % vs. 63.2 % and for ZR-75-1 86.8 % vs. 82.9 %, respectively. The cell motility including 2D movement and 3D transmembrane migration were higher after quick thawing at 100 °C. Invasive abilities of cells after cryopreservation were higher than that of fresh (non-treated cells). Both thawing regimes showed a similar rate of cell proliferation. Cryopreservation procedures, and especially this one with quick thawing, increase malignancy of ZR-75-1 and MDA-MB-231 breast cancer cells and risk of metastasis.

Extrusion of Neutrophil Extracellular Traps (NETs) Negatively Impacts Canine Sperm Functions: Implications in Reproductive Failure.

Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.

Sustainable Production of Biofuels and Biochemicals via Electro-Fermentation Technology.

The energy crisis and climate change are two of the most concerning issues for human beings nowadays. For that reason, the scientific community is focused on the search for alternative biofuels to conventional fossil fuels as well as the development of sustainable processes to develop a circular economy. Bioelectrochemical processes have been demonstrated to be useful for producing bioenergy and value-added products from several types of waste. Electro-fermentation has gained great attention in the last few years due to its potential contribution to biofuel and biochemical production, e.g., hydrogen, methane, biopolymers, etc. Conventional fermentation processes pose several limitations in terms of their practical and economic feasibility. The introduction of two electrodes in a bioreactor allows the regulation of redox instabilities that occur in conventional fermentation, boosting the overall process towards a high biomass yield and enhanced product formation. In this regard, key parameters such as the type of culture, the nature of the electrodes as well as the operating conditions are crucial in order to maximize the production of biofuels and biochemicals via electro-fermentation technology. This article comprises a critical overview of the benefits and limitations of this emerging bio-electrochemical technology and its contribution to the circular economy.

Natural outbreak of Mycobacterium caprae infection in imported laboratory cynomolgus macaques (Macaca fascicularis): diagnostic pitfalls and management of safety precautions.

Tuberculosis (TB) is a major health threat for humans and for non-human primates used for toxicology or research purposes. Emerging mycobacterial species represent a major challenge for diagnosis and surveillance programs. Here, we report a natural outbreak of Mycobacterium caprae in imported cynomolgus macaques (Macaca fascicularis) that occurred at AnaPath Research S.A.U. (APR). The macaques underwent repeated negative intradermal tuberculin tests (IDT) before importation and at the European quarantine station. Exhaustive TB screening was started at APR after confirmation of one positive case at another facility. The animal in question belonged to the same colony received at APR. Diagnostic approaches included clinical examination, PCR, culture, spoligotyping, IDT testing, interferon-γ release assay (IGRA), and thoracoabdominal ultrasound (US). Three regulatory toxicity studies and stock animals were affected. The macaques lacked clinical signs, except for one showing a fistulizing nodule in the right inguinal area, which tested positive for the Mycobacterium tuberculosis complex by PCR. All animals were necropsied and 10 macaques (n=114) showed gross and histologic findings compatible with TB confirmed by PCR and culture. M. caprae was identified as the etiological agent by Direct Variable Repeat spacer oligonucleotide typing (DVR spoligotyping). The infection was traced to Asia via the SB1622 spoligotype involved, confirming that the animals were infected prior to their import into Europe. Tuberculin skin test (TST), IGRA, and US were only sensitive in detecting advanced cases of M. caprae infection. One staff member showed a positive TST reaction, which was handled in accordance with the Spanish government's health regulations. All the sanitary measures implemented were effective in eradicating the disease.

HFIP as a versatile solvent in resorcin[n]arene synthesis.

Herein, we present 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) as an efficient solvent for synthesizing resorcin[n]arenes in the presence of catalytic amounts of HCl at ambient temperature and within minutes. Remarkably, resorcinols with electron-withdrawing groups and halogens, which are reported in the literature as the most challenging precursors in this cyclization, are tolerated. This method leads to a variety of 2-substituted resorcin[n]arenes in a single synthetic step with isolated yields up to 98%.

The pig is a better model than the rabbit or rat for studying the pathophysiology of human mesenteric arteries.

AIMS: Animal models are essential to investigate cardiovascular pathophysiology and pharmacology, but phylogenetic diversity makes it necessary to identify the model with vasculature most similar to that of humans. METHODS AND RESULTS: In this study, we compared the mesenteric arteries of humans, pigs, rabbits and rats in terms of the i) evolutionary changes in the amino acid sequences of α1 and ß2 adrenoceptors; M1, M2, and M3 muscarinic receptors; and bradykinin (BKR) and thromboxane-prostanoid (TP) receptors, through bioinformatics tools; ii) expression of α1, ß2, M1, M3 and TP receptors in each tunica, as assessed by immunofluorescence; and iii) reactivity to receptor-dependent and independent contractile agonists and relaxants, by performing organ bath assays. Phylogenetically, pigs showed the highest degree of evolutionary closeness to humans for all receptors, and with the exception of BKR, rabbits presented the greatest evolutionary difference compared to humans, pigs and rats. The expression of the measured receptors in the three vascular tunica in pigs was most similar to that in humans. Using a one-way ANOVA to determine the differences in vascular reactivity, we found that the reactivity of pigs was the most similar to that of humans in terms of sensitivity (pD2) and maximum effect of vascular reactivity (Emax) to KCl, phenylephrine, isoproterenol and carbachol. CONCLUSIONS: The pig is a better vascular model than the rabbit or rat to extrapolate results to human mesenteric arteries. Comparative vascular studies have implications for understanding the evolutionary history of different species. TRANSLATIONAL PERSPECTIVE: The presented findings are useful for identifying an animal model with a vasculature that is similar to that of humans. This information is important to extrapolate, with greater precision, the findings in arterial pathophysiology or pharmacology from animal models to the healthy or diseased human being.

Vascular adaptation to cancer beyond angiogenesis: The role of PTEN.

Cancer is a public health problem, and it needs blood vessels to grow. Knowing more about the processes of vascular adaptation to cancer improves our chances of attacking it, since the tumor for its extension needs such adaptation to satisfy its progressive demand for nutrients. The main objective of this review is to present the reader with some fundamental molecular pathways for vascular adaptation to cancer, highlighting within them the regulatory role of homologous tensin and phosphatase protein (PTEN). Hence the review describes vascular adaptation to cancer through somewhat known processes, such as angiogenesis, but emphasizes others that are much less explored, namely the changes in vascular reactivity and remodeling of the vascular wall -intima-media thickness and adjustments in the extracellular matrix- The role of PTEN in physiological and pathological vascular mechanisms in different types of cancer is deepened, as a crucial mediator in vascular adaptation to cancer, and points pending further exploration in cancer vascularization are suggested.

In vivo absolute quantification of carnosine in the vastus lateralis muscle with 1H MRS using a surface coil and water as internal reference.

OBJECTIVE: To standardize a method for 1H MRS intramuscular absolute quantification of carnosine in the thigh, using a surface coil and water as internal reference. MATERIALS AND METHODS: Carnosine spectra were acquired in phantoms (5, 10, and 15 mM) as well as in the right gastrocnemius medialis (GM) and right vastus lateralis (VLM) muscles of young team sports athletes, using volume (VC) and surface (SC) coils on a 3 T scanner, with the same receiver gain. Water spectra were used as internal reference for the absolute quantification of carnosine. RESULTS: Phantom's experiments showed a maximum error of 7%, highlighting the validity of the measurements in the study setup. The carnosine concentrations (mmol/kg ww, mean ± SD) measured in the GM were 6.8 ± 2.2 with the VC (CcarVC) and 10.2 ± 3.0 with the SC (CcarSC) (P = 0.013; n = 9). Therefore, a correction was applied to these measurements (CcarVC = 0.6582*CcarSC), to make coils performance comparable (6.8 ± 2.2 for VC and 6.7 ± 2.0 for SC, P = 0.97). After that, only the SC was used to quantify carnosine in the VLM, where a concentration of 5.4 ± 1.5 (n = 30) was found, with significant differences between men (6.2 ± 1.3; n = 15) and women (4.6 ± 1.2; n = 15). The error in quantitation was 5.3-5.5% with both coils. CONCLUSION: The method using the SC and water as internal reference can be used to quantify carnosine in voluminous muscles and regions of the body in humans, where the VC is not suitable, such as the VLM.

Neurological Pupil Index and Delayed Cerebral Ischemia after Subarachnoid Hemorrhage: A Retrospective Multicentric Study.

BACKGROUND: Delayed cerebral ischemia (DCI) occurs in around 30% of patients suffering from nontraumatic subarachnoid hemorrhage (SAH) and is associated with poor neurological outcome. Whether the Neurological Pupil index (NPi) derived from the automated pupillometry could help to diagnose the occurrence of DCI remains unknown. The aim of this study was to investigate the association of NPi with the occurrence of DCI in patients with SAH. METHODS: This was a multicenter, retrospective cohort study of consecutive patients with SAH admitted to the intensive care units of five hospitals between January 2018 and December 2020 who underwent daily NPi recordings (every 8 h) during the first 10 days of admission. DCI was diagnosed according to standard definitions (in awake patients) or based on neuroimaging and neuromonitoring (in sedated or unconscious patients). An NPi < 3 was defined as abnormal. The primary outcome of the study was to assess the time course of daily NPi between patients with DCI and patients without DCI. Secondary outcome included the number of patients who had an NPi < 3 before DCI. RESULTS: A total of 210 patients were eligible for the final analysis; DCI occurred in 85 (41%) patients. Patients who developed DCI had similar values of mean and worst daily NPi over time when compared with patients without DCI. Patients with DCI had a higher proportion of at least one NPi < 3 at any moment before DCI when compared with others (39/85, 46% vs. 35/125, 38%, p = 0.009). Similarly, the worst NPi before DCI diagnosis was lower in the DCI group when compared with others (3.1 [2.5-3.8] vs. 3.7 [2.7-4.1], p = 0.05). In the multivariable logistic regression analysis, the presence of NPi < 3 was not independently associated with the development of DCI (odds ratio 1.52 [95% confidence interval 0.80-2.88]). CONCLUSIONS: In this study, NPi measured three times a day and derived from the automated pupillometry had a limited value for the diagnosis of DCI in patients with SAH.

Development of a Frugal, In Situ Sensor Implementing a Ratiometric Method for Continuous Monitoring of Turbidity in Natural Waters.

Turbidity is a commonly used indicator of water quality in continental and marine waters and is mostly caused by suspended and colloidal particles such as organic and inorganic particles. Many methods are available for the measurement of turbidity, ranging from the Secchi disk to infrared light-based benchtop or in situ turbidimeters as well as acoustic methods. The operational methodologies of the large majority of turbidity instruments involve the physics of light scattering and absorption by suspended particles when light is passed through a sample. As such, in the case of in situ monitoring in water bodies, the measurement of turbidity is highly influenced by external light and biofouling. Our motivation for this project is to propose an open-source, low-cost in situ turbidity sensor with a suitable sensitivity and operating range to operate in low-to-medium-turbidity natural waters. This prototype device combines two angular photodetectors and two infrared light sources with different positions, resulting in two different types of light detection, namely nephelometric (i.e., scattering) and attenuation light, according to the ISO 7027 method. The mechanical design involves 3D-printed parts by stereolithography, which are compatible with commercially available waterproof enclosures, thus ensuring easy integration for future users. An effort was made to rely on mostly off-the-shelf electronic components to encourage replication of the system, with the use of a highly integrated photometric front-end commonly used in portable photoplethysmography systems. The sensor was tested in laboratory conditions against a commercial benchtop turbidimeter with Formazin standards. The monitoring results were analyzed, obtaining a linear trendline from 0 to 50 Nephelometric Turbidity Unit (NTU) and an accuracy of +/-0.4 NTU in the 0 to 10 NTU range with a response time of less than 100 ms.

Comparative Transcriptomic Analyses for the Optimization of Thawing Regimes during Conventional Cryopreservation of Mature and Immature Human Testicular Tissue.

Cryopreservation of human testicular tissue, as a key element of anticancer therapy, includes the following stages: saturation with cryoprotectants, freezing, thawing, and removal of cryoprotectants. According to the point of view existing in "classical" cryobiology, the thawing mode is the most important consideration in the entire process of cryopreservation of any type of cells, including cells of testicular tissue. The existing postulate in cryobiology states that any frozen types of cells must be thawed as quickly as possible. The technologically maximum possible thawing temperature is 100 °C, which is used in our technology for the cryopreservation of testicular tissue. However, there are other points of view on the rate of cell thawing, according to how thawing should be carried out at physiological temperatures. In fact, there are morphological and functional differences between immature (from prepubertal patients) and mature testicular tissue. Accordingly, the question of the influence of thawing temperature on both types of tissues is relevant. The purpose of this study is to explore the transcriptomic differences of cryopreserved mature and immature testicular tissue subjected to different thawing methods by RNA sequencing. Collected and frozen testicular tissue samples were divided into four groups: quickly (in boiling water at 100 °C) thawed cryopreserved mature testicular tissue (group 1), slowly (by a physiological temperature of 37 °C) thawed mature testicular tissue (group 2), quickly thawed immature testicular tissue (group 3), and slowly thawed immature testicular tissue (group 4). Transcriptomic differences were assessed using differentially expressed genes (DEG), the Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and protein-protein interaction (PPI) analyses. No fundamental differences in the quality of cells of mature and immature testicular tissue after cryopreservation were found. Generally, thawing of mature and immature testicular tissue was more effective at 100 °C. The greatest difference in the intensity of gene expression was observed in ribosomes of cells thawed at 100 °C in comparison with cells thawed at 37 °C. In conclusion, an elevated speed of thawing is beneficial for frozen testicular tissue.

Subaquatic indirect laser ablation technique for glass processing.

Subaquatic indirect Laser-Induced Plasma-Assisted Ablation (SLIPAA) is proposed as a laser-based technique for glass processing. In this configuration, a water layer is added between a metallic target and a soda-lime glass substrate, so the processing of the glass is due to a combination of the ablation mechanism, the shock waves, and the cavitation bubbles. Thus, this method makes it possible to produce higher depth structures than those performed up to now by other standard laser techniques based on ablation, achieving structures in glass with rectangular cross-sectional profiles. Channels of 1 mm width are fabricated, reaching an average maximal depth value of almost 1400 µm at 30 passes of the laser beam while keeping the focal position fixed. Furthermore, the difference between processing the material with and without the addition of the water layer is presented. The influence of the processing parameters on the shape and quality of the fabricated structures is studied by optical and confocal microscopy, microcomputed tomography, and scanning electron microscopy. Compositional analysis of the glass is performed by energy dispersive X-ray technique to assess the transference of material from the metallic target to the fabricated channels. Deeper and more complex structures are obtained by refocusing the laser beam on the target and adding a pulsed flowing water film.

Autophagy is activated in human spermatozoa subjected to oxidative stress and its inhibition impairs sperm quality and promotes cell death.

STUDY QUESTION: Does oxidative stress (OS) activate autophagy in human sperm? SUMMARY ANSWER: Human spermatozoa subjected to OS activate an autophagic response. WHAT IS KNOWN ALREADY: Autophagy is a regulated pathway of lysosomal degradation which helps eukaryotic cells to maintain or restore homeostasis, being a cellular stress response mechanism. OS is a main cause of impaired sperm function and is linked to male infertility; however, whether OS activates autophagy in human spermatozoa is unknown. STUDY DESIGN, SIZE, DURATION: Human spermatozoa were exposed separately to ionomycin and hydrogen peroxide in order to induce OS. An untreated control group was included. Sperm cells under OS were then exposed to chloroquine in order to block autophagy. An untreated control and a control incubated only with the OS inducer were included in each experimental setting. PARTICIPANTS/MATERIALS, SETTING, METHODS: For this study, semen samples from normozoospermic donors were used and motile sperm cells were selected by the swim up technique. First, the generation of OS under our experimental conditions was demonstrated by analyzing sperm parameters including viability, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) motility and thiol oxidation. Then, proteins involved in autophagy, including the microtubule-associated protein light chain 3 (LC3), particularly LC3-I and LC3-II, autophagy-related 5 (ATG5) and autophagy-related 16 (ATG16) proteins as well as the phosphorylated form of AMP-activated protein kinase (pAMPK) were evaluated in spermatozoa exposed to OS and compared to the untreated control. Finally, the impact of autophagy blocking by chloroquine treatment on sperm quality, metabolic parameters, including glycolysis and oxidative phosphorylation, as well as the cell death markers phosphatidylserine externalization and caspase activation was analyzed. Sperm quality parameters, cell death markers and autophagy-related proteins were analyzed by flow cytometry. Motility was evaluated by the computer-assisted sperm analysis system and metabolic parameters were analyzed using an extracellular flux analyzer. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to ionomycin and hydrogen peroxide promotes OS resulting in increased ROS production and decreased viability, ΔΨm and motility, while increasing thiol oxidation. These alterations were accompanied by a decrease in LC3-I, indicating that autophagy was activated upon OS exposure. Ionomycin also caused an increase in LC3-II, ATG5, ATG16 and pAMPK content. Autophagy blocking of sperm exposed to OS caused deterioration in sperm quality and metabolic parameters as well as an increase in cell death markers. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from normozoospermic donors; tests on sperm from infertile patients were not carried out. The autophagy blocking plus OS might generate a non-specific response to a highly stressful situation leading to the induction of cell death. WIDER IMPLICATIONS OF THE FINDINGS: Human spermatozoa subjected to OS activate an autophagic response and its blockage results in increased oxidative damage and commits spermatozoa to cell death. These results suggest a crucial role of autophagy as a stress response by male gametes, which contributes to maintaining the functionality and lifespan of ejaculated sperm cells. Detection of autophagy activation in sperm cells ex vivo could be included in semen analysis as a marker of OS, especially in men displaying high levels of seminal ROS. Novel strategies that aim to activate this cellular stress response could improve sperm quality/functionality under natural ejaculate conditions in which increased ROS levels are expected. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Fondo Nacional de Investigación Científica y Tecnológica, Chile (ANID/FONDECYT, Grant number 11170758 to P.U.); the Comisión Nacional de Investigación Científica y Tecnológica, Chile (ANID/CONICYT, Grant number PAI79160030 to P.U.) and the Dirección de Investigación, Universidad de La Frontera. The authors disclose no potential conflicts of interest.

Efficacy of high-intensity interval- or continuous aerobic-training on insulin resistance and muscle function in adults with metabolic syndrome: a clinical trial.

PURPOSE: We carried out a randomized, clinical trial in adults of both sexes with metabolic syndrome (MS) to assess the efficacy of high-intensity, low-volume interval training (HIIT) compared to moderate-intensity continuous training (MICT) on insulin resistance (IR), muscle mass, muscle activation, and serum musclin. METHODS: Fasting glycemia, insulinemia, and glycated haemoglobin were determined by conventional methods, IR by Homeostatic model assessment (HOMA), lean mass by Dual-Energy X-ray Absorptiometry, muscle activation through carnosine by Proton Magnetic Resonance Spectroscopy, and musclin by Enzyme-Linked ImmunoSorbent Assay before and after a supervised, three-times/week, 12-week treadmill programme. HIIT (n = 29) consisted of six intervals with one-minute, high-intensity phases at 90% of peak oxygen consumption (VO2peak). MICT (n = 31) trained at 60% of VO2peak for 30 min. RESULTS: Patients had a mean age of 50.8 ± 6.0 years, body mass index of 30.6 ± 4.0 kg/m2, and VO2peak of 29.0 ± 6.3 mL.kg-1.min-1. Compared to MICT, HIIT was not superior at reducing Ln HOMA-IR (adjusted mean difference: 0.083 [95%CI - 0.092 to 0.257]), carnosine or musclin or at increasing thigh lean mass. HIIT increased carnosine by 0.66 mmol/kg.ww (95% CI 0.08-1.24) after intervention. Both interventions reduced IR, body fat percentage and increased total lean mass/height2 and VO2peak. Musclin showed a non-significant reduction with a small effect size after both interventions. CONCLUSION: Compared to MICT, HIIT is not superior at reducing IR, carnosine or musclin or at increasing skeletal muscle mass in adults with MS. Both training types improved IR, muscle mass and body composition. NCT03087721, March 22nd, 2017. TRIAL REGISTRATION NUMBER: NCT03087721. Registered March 22nd, 2017.

Exposing the inadequacy of redox formalisms by resolving redox inequivalence within isovalent clusters.

In this report we examine a family of trinuclear iron complexes by multiple-wavelength, anomalous diffraction (MAD) to explore the redox load distribution within cluster materials by the free refinement of atomic scattering factors. Several effects were explored that can impact atomic scattering factors within clusters, including 1) metal atom primary coordination sphere, 2) M-M bonding, and 3) redox delocalization in formally mixed-valent species. Complexes were investigated which vary from highly symmetric to fully asymmetric by 57Fe Mössbauer and X-ray diffraction to explore the relationship between MAD-derived data and the data available from these widely used characterization techniques. The compounds examined include the all-ferrous clusters [ n Bu4N][(tbsL)Fe3(µ3-Cl)] (1) ([tbsL]6- = [1,3,5-C6H9(NC6H4-o-NSi t BuMe2)3]6-]), (tbsL)Fe3(py) (2), [K(C222)]2[(tbsL)Fe3(µ3-NPh)] (4) (C222 = 2,2,2-cryptand), and the mixed-valent (tbsL)Fe3(µ3-NPh) (3). Redox delocalization in mixed-valent 3 was explored with cyclic voltammetry (CV), zero-field 57Fe Mössbauer, near-infrared (NIR) spectroscopy, and X-ray crystallography techniques. We find that the MAD results show an excellent correspondence to 57Fe Mössbauer data; yet also can distinguish between subtle changes in local coordination geometries where Mössbauer cannot. Differences within aggregate oxidation levels are evident by systematic shifts of scattering factor envelopes to increasingly higher energies. However, distinguishing local oxidation levels in iso- or mixed-valent materials can be dramatically obscured by the degree of covalent intracore bonding. MAD-derived atomic scattering factor data emphasize in-edge features that are often difficult to analyze by X-ray absorption near edge spectroscopy (XANES). Thus, relative oxidation levels within the cluster were most reliably ascertained from comparing the entire envelope of the atomic scattering factor data.