White blood cells, TNF-α, and interleukin-6 in subjects with infantile colic treated with Lacticaseibacillus rhamnosus GG (ATCC 53103): a randomised prospective study.

Recent metanalysis reported that certain probiotic strains, such as Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus (LGG), seem effective for treatment of infantile colic of exclusively breastfed infants; some reports have also linked probiotics to have an immunological effect, however further investigation are needed to fully understand the exact mechanism. The objective of this study was to assay white blood cells, tumour necrosis factor (TNF)-α and interleukin (IL)-6 values in peripheral blood in subjects treated in a randomised, double-blind, placebo-controlled trial for infantile colic with LGG. Fifty-eight infants were enrolled and followed for a study period of 28 days. Parent were asked to record daily crying time using a structured cry diary. Peripheral white blood cells was assessed and RNA (mRNA) expression of TNF-α and IL-6 was measured using TaqMan real-time PCR-maternal amplification. Infants with colic treated with LGG showed a reduction in daily crying duration after 28 days of treatment and a reduction in values of IL-6 ( P < 0.005) and TNF-α ( P < 0.05); we observe also a significantly decreasing of IL-6 in the placebo group while decrease of TNF-α was not significant in this group. A significant decreased values of monocytes ( P < 0.05) was observed in infants treated with LGG. Our data therefore showed, in addition to crying time reduction, a significant decrease of TNF-α and a significant reduction of monocytes cells in colicky infants treated with LGG, compared to placebo group. This observation supports the hypothesis that probiotics may have anti-inflammatory properties. Further studies are needed to better understand the influence of probiotic on immunity cells.

Thymic hyperplasia is accurate to detect new-onset Graves' hyperthyroidism and resolves after restoring euthyroidism.

PURPOSE: Abnormal liver blood tests (ALBTs), neutropenia (NEU) and thymic hyperplasia (TH) are new features of Graves' disease (GD). Our objectives were: (a) to calculate the accuracy of TH in discriminating between Graves' and non-Graves' thyrotoxicosis, compared to ALBTs, NEU and Graves' orbitopathy (GO); (b) to explore the outcome of GD-associated TH and non-GD-associated TH. METHODS: We prospectively analyzed consecutive adult patients with newly diagnosed thyrotoxicosis from January 2018 to June 2023. TH was detected via neck ultrasound (nUS) then confirmed and followed by magnetic resonance imaging (MRI). For GD vs non-GD clinical sensitivity (SE) and specificity (SPEC), accuracy, positive predictive value (PPV) and negative predictive value (NPV) of GO, TH, ALBTs and NEU were calculated. RESULTS: 264 thyrotoxic patients were included. TH was found in 16.4% (20/122) of GD vs 1.4% (2/142) in non-GD (p < 0.001). SE, SPEC, accuracy, PPV and NPV of the four extrathyroidal manifestations of GD were as follows, respectively: GO 26%, 100%, 66%, 100%, 61%; ALBTs 41%, 89%, 69%, 76%, 66%; NEU 5%, 100%, 56%, 100%, 55%; TH 16%, 98%, 61%, 91%, 98%. In 18 of them, TH regressed within 12 months after achieving euthyroidism under anti-thyroid drug therapy, while in the remaining 2, TH regressed 6 months after thyroid surgery. In the two non-GD patients with TH, thymus disappeared along with euthyroidism. CONCLUSIONS: TH in the hyperthyroidism scenario provides a high PPV for GD. A conservative approach for the diagnostic work-up and initial management of thyrotoxicosis-associated TH should be adopted.

Tumor enlargement in adrenal incidentaloma is related to glaucoma: a new prognostic feature?

PURPOSE: The uncertainty on the management of small adrenal incidentalomas (AIs) still represents a challenge in real clinical practice. Considering the lack of knowledge on risk factors implicated in tumour enlargement, the aim of this study was to identify risk factors for morphological changes during follow-up of adrenal incidentalomas (AIs). METHODS: We retrospectively evaluated demographic, clinical, radiological and biochemical parameters of 153 AIs (2007-2021). Patients with histological diagnosis of metastases or pheochromocytoma were excluded. To detect risk factors for tumor enlargement, diseases associated with AIs were included if their prevalence was higher than 2%. Patients were divided into two groups (A: radiological stability; B: tumor enlargement defined as > 5 mm/year in the main diameter). RESULTS: Group A: 89.5% and group B: 10.5%, mean follow-up 38.6 ± 6.9 months (range 6-240). Tumor enlargement when occurred was within 36 months of follow-up. In group B high body weight (p < 0.03), dehydroepiandrosterone sulfate (DHEAS) (p < 0.05) and direct renin concentration (DRC) (p < 0.04) were higher than group A, while aldosterone levels were lower; moreover, considering comorbidities, glaucoma and dysglycemia (p < 0.01 for both) had higher prevalence in group B. Glaucoma and dysglycemia were independent predictors of enlargement. Patients affected by glaucoma, atrial fibrillation, dysglycemia had a lower dimensional change-free survival than non-affected. CONCLUSIONS: Glaucoma might be a novel risk factor for AI enlargement. If subtle undetectable cortisol hypersecretion has a role is a topic for further research.

Neuroinflammation and status epilepticus: a narrative review unraveling a complex interplay.

Status epilepticus (SE) is a medical emergency resulting from the failure of the mechanisms involved in seizure termination or from the initiation of pathways involved in abnormally prolonged seizures, potentially leading to long-term consequences, including neuronal death and impaired neuronal networks. It can eventually evolve to refractory status epilepticus (RSE), in which the administration of a benzodiazepine and another anti-seizure medications (ASMs) had been ineffective, and super-refractory status epilepticus (SRSE), which persists for more than 24 h after the administration of general anesthesia. Objective of the present review is to highlight the link between inflammation and SE. Several preclinical and clinical studies have shown that neuroinflammation can contribute to seizure onset and recurrence by increasing neuronal excitability. Notably, microglia and astrocytes can promote neuroinflammation and seizure susceptibility. In fact, inflammatory mediators released by glial cells might enhance neuronal excitation and cause drug resistance and seizure recurrence. Understanding the molecular mechanisms of neuroinflammation could be crucial for improving SE treatment, wich is currently mainly addressed with benzodiazepines and eventually phenytoin, valproic acid, or levetiracetam. IL-1ß signal blockade with Anakinra has shown promising results in avoiding seizure recurrence and generalization in inflammatory refractory epilepsy. Inhibiting the IL-1ß converting enzyme (ICE)/caspase-1 is also being investigated as a possible target for managing drug-resistant epilepsies. Targeting the ATP-P2X7R signal, which activates the NLRP3 inflammasome and triggers inflammatory molecule release, is another avenue of research. Interestingly, astaxanthin has shown promise in attenuating neuroinflammation in SE by inhibiting the ATP-P2X7R signal. Furthermore, IL-6 blockade using tocilizumab has been effective in RSE and in reducing seizures in patients with febrile infection-related epilepsy syndrome (FIRES). Other potential approaches include the ketogenic diet, which may modulate pro-inflammatory cytokine production, and the use of cannabidiol (CBD), which has demonstrated antiepileptic, neuroprotective, and anti-inflammatory properties, and targeting HMGB1-TLR4 axis. Clinical experience with anti-cytokine agents such as Anakinra and Tocilizumab in SE is currently limited, although promising. Nonetheless, Etanercept and Rituximab have shown efficacy only in specific etiologies of SE, such as autoimmune encephalitis. Overall, targeting inflammatory pathways and cytokines shows potential as an innovative therapeutic option for drug-resistant epilepsies and SE, providing the chance of directly addressing its underlying mechanisms, rather than solely focusing on symptom control.

Sensitivity of PM10 oxidative potential to aerosol chemical composition at a Mediterranean urban site: ascorbic acid versus dithiothreitol measurements.

The oxidative potential (OP) of PM10 daily samples collected at a traffic site in southeastern Spain during summer and winter was assessed by two acellular assays: the ascorbic acid (AA) and dithiothreitol (DTT) methods. Although PM10 levels were similar during both periods, OP values (expressed in nmol min-1 m-3) showed a defined seasonal trend. The AA activity was higher in summer than in winter, whereas the DTT reactivity exhibited an opposite seasonal pattern. Both assays were sensitive to different PM10 components, as shown by the results of the linear correlation analysis. Moreover, the relationship between OP values and PM10 chemical species was not the same during summer and winter, indicating that particle toxicity is associated with different sources during the warm and cold seasons. When OP values were expressed on a mass basis (nmol min-1 µg-1), lower correlation coefficients with PM10 chemical species were generally obtained compared to volume-normalized activities. These outcomes suggest that only some specific components have a significant intrinsic oxidative potential. Supplementary Information: The online version contains supplementary material available at 10.1007/s11869-023-01332-1.

Combination Blockade of the IL6R/STAT-3 Axis with TIGIT and Its Impact on the Functional Activity of NK Cells against Prostate Cancer Cells.

Methods: We analyzed the secretion of cytokines, chemokines, and growth factors in 22Rv1, LNCaP, and DU145 cells. In these cells, we also evaluated the expression of NK ligands, IL6R, STAT-3, and phosporylated STAT-3. In NK-92 cells, we evaluated the effects of Stattic (Stt) and tocilizumab (Tcz) on NK receptors. In addition, we assessed if the disruption of the IL6R/STAT-3 pathway and blockade of TIGIT potentiated the cytotoxicity of NK-92 cells versus DU145 cells. Results: DU145 abundantly secretes M-CSF, VEGF, IL-6, CXCL8, and TGF-ß. Furthermore, the expression of CD155 was found to increase in accordance with aggressiveness and metastatic status in the prostate cancer cells. Stt and Tcz induce a decrease in STAT-3 phosphorylation in the DU145 cells and, in turn, induce an increase of NKp46 and a decrease of TIGIT expression in NK-92 cells. Finally, the disruption of the IL6R/STAT-3 axis in prostate cancer cells and the blocking of TIGIT on NK-92 were observed to increase the cytotoxicity of NK-92 cells against DU145 cells through an increase in sFasL, granzyme A, granzyme B, and granulysin. Conclusions: Our results reveal that the combined use of inhibitors directed against the IL6R/STAT-3 axis and TIGIT enhances the functional activity of NK cells against castration-resistant prostate cancer cells.

Size segregated ionic species collected in a harbour area.

Water-soluble ions were analysed in size segregated aerosol samples collected in the port of Alicante (Southeastern Spain) during summer and winter using a multistage cascade impactor. Seasonal variations in the size distributions of the analysed components and the influence of bulk materials handling (loading/unloading and stockpiling) at the docks were investigated. The size distributions of SO42-, NH4+ and K+ were characterized by prominent peaks in the condensation and droplet modes, both in summer and winter, while those of Ca2+, Na+, Mg2+ and Cl- had a main peak centred at ∼4 µm. Although oxalate size distributions were similar during both seasons, the fraction of coarse-mode oxalate increased in summer most likely as a result of volatilization and repartition processes or reactions of oxalic acid with coarse alkaline particles. Nitrate size distributions were dominated by a coarse mode; however, during winter, modal peaks in the submicron size range were also observed due to favourable conditions for the formation of fine-mode NH4NO3. Harbour activities had a significant impact only on the concentrations of calcium, particularly in the coarse fraction, during both summer and winter.

The MELD Score Is Superior to the Maddrey Discriminant Function Score to Predict Short-Term Mortality in Alcohol-Associated Hepatitis: A Global Study.

INTRODUCTION: Several scoring systems predict mortality in alcohol-associated hepatitis (AH), including the Maddrey discriminant function (mDF) and model for end-stage liver disease (MELD) score developed in the United States, Glasgow alcoholic hepatitis score in the United Kingdom, and age, bilirubin, international normalized ratio, and creatinine score in Spain. To date, no global studies have examined the utility of these scores, nor has the MELD-sodium been evaluated for outcome prediction in AH. In this study, we assessed the accuracy of different scores to predict short-term mortality in AH and investigated additional factors to improve mortality prediction. METHODS: Patients admitted to hospital with a definite or probable AH were recruited by 85 tertiary centers in 11 countries and across 3 continents. Baseline demographic and laboratory variables were obtained. The primary outcome was all-cause mortality at 28 and 90 days. RESULTS: In total, 3,101 patients were eligible for inclusion. After exclusions (n = 520), 2,581 patients were enrolled (74.4% male, median age 48 years, interquartile range 40.9-55.0 years). The median MELD score was 23.5 (interquartile range 20.5-27.8). Mortality at 28 and 90 days was 20% and 30.9%, respectively. The area under the receiver operating characteristic curve for 28-day mortality ranged from 0.776 for MELD-sodium to 0.701 for mDF, and for 90-day mortality, it ranged from 0.773 for MELD to 0.709 for mDF. The area under the receiver operating characteristic curve for mDF to predict death was significantly lower than all other scores. Age added to MELD obtained only a small improvement of AUC. DISCUSSION: These results suggest that the mDF score should no longer be used to assess AH's prognosis. The MELD score has the best performance in predicting short-term mortality.

Quantification of the impact of port activities on PM10 levels at the port-city boundary of a mediterranean city.

The main objective of this work was to quantify the impact of handling of bulk materials on PM10 levels measured at the port-city border of Alicante (Spain), located on the western Mediterranean coast. To achieve that goal, 355 PM10 samples were collected at the perimeter of the harbor of Alicante from March 2017 to February 2018. A 181 sample subgroup was chemically characterized in order to perform a source apportionment study with the EPA PMF 5.0 model. Eight factors were identified, two of them directly related to the handling of bulk materials (Limestone + gypsum and Clinker), accounting jointly for 35% of the average PM10 concentration. A Road traffic factor was the second highest contributor to PM10 levels (17%) while the Shipping emissions factor accounted for only 6% of the average PM10 mass. Other factors such as Biomass burning+ secondary nitrate and Aged sea salt represented a joint contribution of 25% of the PM10 mass. Results indicate that emission abatement strategies should primarily focus on the reduction of fugitive emissions caused by the handling of bulk materials at the docks. Moreover, scenarios including reductions of more than 50% in bulk handling sources and 10% in other anthropogenic sources would help to reduce anthropogenic exceedances of the daily PM10 limit (50 µg·m-3) and to approach to WHO daily PM10 standard (20 µg m-3).

A 1000-year-old mystery solved: Unlocking the molecular structure for the medieval blue from Chrozophora tinctoria, also known as folium.

The molecular structure of the medieval watercolor known as folium has finally been solved in the 21st century. The interdisciplinary approach taken was the key to producing extracts that had been prepared following medieval instructions, and shows the blue/purple chromophore as the major dye in Chrozophora tinctoria fruits (shell). A multi-analytical characterization of its structure was made using HPLC-DAD-MS, GC-MS, NMR (1H, 13C, COSY, HSQC, HMBC, INADEQUATE), and computational studies. The results demonstrate that the blue compound corresponds to 6'-hydroxy-4,4'-dimethoxy-1,1'-dimethyl-5'-{[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]oxy}-[3,3'-bipyridine]-2,2',5,6(1H,1'H)-tetraone, a hermidin derivative, which we named chrozophoridin. Experimental data and computational modeling studies show that this mono-glycosylated dimer is represented by two stable isomers (atropisomers). This is an indispensable piece of knowledge for the characterization of this medieval dye in works of art such as medieval manuscript illuminations and for testing its stability and contributes to the preservation of our cultural heritage.

HPLC-MS/MS measurement of lidocaine in rat skin and plasma. Application to study the release from medicated plaster.

A simple, sensitive HPLC-MS/MS method was developed and validated for the determination of lidocaine in skin and plasma of rats. The methods were established and validated assessing lower limit of quantitation (LLOQ), linearity, intra and inter-day precision and accuracy, selectivity, recovery and matrix effect. Chromatography was done on a Gemini column embedded with C18 stationary phase (50 mm × 2.0 mm, 5 µm particle size), using a gradient with mobile phases consisting of 0.1% HCOOH in bidistilled water and 0.1% HCOOH in acetonitrile. The mass spectrometer worked with electrospray ionization in positive ion mode and selected reaction monitoring, using target ions m/z 235.10 for lidocaine and m/z 245.10 for lidocaine-d10, used as internal standard. RESULTS: The linearity of the method was in the ranges of lidocaine concentrations 10.0-200.0 ng/mL for skin homogenate (accuracy 94.1-105.5%; R2 ≥ 0.998) and 0.025-2 ng/mL for plasma (accuracy 96.2-104.8%; R2 ≥ 0.996). The intra- and inter-day precision and accuracy determined on three quality control samples (20, 75 and 170 ng/mL for skin and 0.075, 0.4 and 1.5 ng/mL for plasma) were ≤4.2% and 103.8-108.2% for skin and ≤12.4% and 95.5-101.4% for plasma. The LLOQ was 10 ng/mL in skin homogenate and 0.025 ng/mL in plasma. The applicability of the method was demonstrated by measuring lidocaine in skin and plasma after exposure to medicated patches containing 5% lidocaine.

Insights into the origin and evolution of carbonaceous aerosols in a mediterranean urban environment.

Organic carbon (OC) and elemental carbon (EC) concentrations were measured in PM1 and PM10 daily samples collected at an urban station in Elche (southeastern Spain) from February 2015 to February 2018. The effect of seasonal weather conditions, traffic, and specific pollution events (Saharan dust outbreaks and local pollution episodes) on the variability of carbonaceous aerosol levels was studied in this work. The joint contribution of carbonaceous species to PM1 and PM10 mass concentrations was, respectively, 48% and 26%. Both OC and EC concentrations were higher in winter than in summer because of the poor dispersion conditions and lower temperatures leading to the condensation of semivolatile species. Secondary organic carbon (SOC), estimated using the EC tracer method, also exhibited higher concentrations during winter, indicating that the prevailing meteorological conditions during the cold season are more favorable for the formation of secondary organic aerosols. Our results suggest different formation pathways of secondary organic components during summer and winter. At the sampling site, EC was primarily derived from traffic emissions, independently of the season and the type of event, with a modest contribution from biomass burning (<20%). The estimated contribution from this source to OC levels was similar. Local pollution episodes lead to a significant increase in the concentrations of carbonaceous species, in particular of SOC, influencing its temporal variation. On average, African dust outbreaks showed a moderate impact on the levels of carbonaceous aerosols; however, the effect was significantly stronger during winter Saharan events.

Cojera no traumática en Urgencias de Pediatría. Epidemiología, valoración y resultados / Non-traumatic limping in Paediatric Emergencies: Epidemiology, evaluation and results

Introducción. La cojera no traumática es un motivo frecuente de consulta en urgencias pediátricas (UPED). Aunque el diagnóstico más frecuente es la sinovitis transitoria de cadera (STC), existen cojeras secundarias a patologías graves. El objetivo de este estudio es describir las variables relacionadas con las cojeras no traumáticas en UPED, las características clínicas, las pruebas complementarias y el diagnóstico final para establecer el mejor protocolo de manejo, rentabilizando recursos y agilizando la asistencia en urgencias. Material y método. Estudio prospectivo, en niños<15 años que consultaron en UPED por cojera no traumática durante el año 2014. Se recogieron variables clínicas, exploraciones complementarias, diagnóstico en urgencias y a los 6 meses. Resultados. Durante el 2014 se incluyó en el estudio por cojera no traumática a 146 pacientes, el 0,69% de las urgencias atendidas. Se diagnosticaron 4 casos de cojera grave: 2 leucemias, una artritis séptica y un tumor óseo acetabular. El diagnóstico más frecuente fue la STC (57,53%). A los 6 meses, 135 niños (92,4%) habían resuelto la cojera. Se modificó el diagnóstico en 9 pacientes (6,1%). Los niños con STC presentaban menos días de evolución y el 77% tenían entre 3 y 10 años. Los niños con diagnóstico final de cojera grave presentaban una cojera más prolongada, fiebre y no apoyo en la deambulación. Conclusiones. En las cojeras con probable origen en la cadera en niños entre 3 años y 10 años, con ausencia de fiebre o síntomas sistémicos y evolución menor de una semana es posible realizar un diagnóstico clínico de STC limitando las exploraciones complementarias (AU)

Introduction. Non-traumatic limping is a common reason for consultation in paediatric emergencies. Although transient synovitis of the hip (TS) is the most frequent diagnosis, there are cases of limping secondary to serious pathologies. The aim of this review is to describe the variables related to non-traumatic limp that come to the paediatric emergency department to establish the best management protocol, making the most of resources and speeding up emergency care. Material and method. A prospective study was conducted, selecting all children less than 15 years old who consulted aspaediatric emergencies for non-traumatic limping during the 2014. Clinical variables, complementary examinations and diagnoses were collected in the emergency room consultation and 6 months after the consultation. Results. During 2014, 146 patients (0.69% of the emergencies) were included in the non-traumatic limping study. Four cases of severe limping were diagnosed: 2 leukaemias, 1 septic arthritis and 1 acetabular bone tumour. The most frequent diagnosis was TS (53.16%). At 6 months, 135 children (92.4%) had resolved lameness. The diagnosis was changed in 9 children (6.1%). Children with TS had fewer days of evolution, and 77% were between 3 and 10 years old. Children with a final diagnosis of severe pathology had a limp for longer, fever and did not weight bear on ambulation. Conclusions. In limping of probable hip origin, at the ages of between 3 and 10, without fever or systemic symptoms and of less than one week's onset, it is possible to make a clinical diagnosis of TS limiting the use of complementary examinations (AU)

In ovo feeding of carbohydrates for broilers-a systematic review.

The purpose of this systematic review was to evaluate the evidence that the injection of carbohydrate-based solutions into embryonated eggs improves broiler performance. A literature search was conducted in April 2017 using the keywords broiler, carbohydrate, in ovo, nutrition and poultry. Only papers that involved in ovo carbohydrate injections in poultry were used in this study. After specific selection criteria, 17 papers were selected. The quality scoring system of the selected studies was based on the injection methodology, use of control groups, type of solution injected, period of injection, egg and hens characteristics, number of variables analysed and the statistical design. Among papers, there was no standardised procedure in to inoculate the solutions. Nevertheless, in general, in ovo feeding of carbohydrates decreases the hatch rate, improves the hatch weight, but it does not seem to influence the post-hatch performance of broilers. The inoculation of 75 mg of glucose in the albumen seems to bring better results. Further studies are needed to improve the technical methodology of in ovo injections for commercial use.

Non-traumatic limping in Paediatric Emergencies: Epidemiology, evaluation and results. / Cojera no traumática en Urgencias de Pediatría. Epidemiología, valoración y resultados.

INTRODUCTION: Non-traumatic limping is a common reason for consultation in paediatric emergencies. Although transient synovitis of the hip (TS) is the most frequent diagnosis, there are cases of limping secondary to serious pathologies. The aim of this review is to describe the variables related to non-traumatic limp that come to the paediatric emergency department to establish the best management protocol, making the most of resources and speeding up emergency care. MATERIAL AND METHOD: A prospective study was conducted, selecting all children less than 15 years old who consulted aspaediatric emergencies for non-traumatic limping during the 2014. Clinical variables, complementary examinations and diagnoses were collected in the emergency room consultation and 6 months after the consultation RESULTS: During 2014, 146 patients (0.69% of the emergencies) were included in the non-traumatic limping study. Four cases of severe limping were diagnosed: 2 leukaemias, 1 septic arthritis and 1 acetabular bone tumour. The most frequent diagnosis was TS (53.16%). At 6 months, 135 children (92.4%) had resolved lameness. The diagnosis was changed in 9 children (6.1%). Children with TS had fewer days of evolution, and 77% were between 3 and 10 years old. Children with a final diagnosis of severe pathology had a limp for longer, fever and did not weight bear on ambulation. CONCLUSIONS: In limping of probable hip origin, at the ages of between 3 and 10, without fever or systemic symptoms and of less than one week's onset, it is possible to make a clinical diagnosis of TS limiting the use of complementary examinations.

Quantitative imaging of anti-phase domains by polarity sensitive orientation mapping using electron backscatter diffraction.

Advanced structural characterisation techniques which are rapid to use, non-destructive and structurally definitive on the nanoscale are in demand, especially for a detailed understanding of extended-defects and their influence on the properties of materials. We have applied the electron backscatter diffraction (EBSD) technique in a scanning electron microscope to non-destructively characterise and quantify antiphase domains (APDs) in GaP thin films grown on different (001) Si substrates with different offcuts. We were able to image and quantify APDs by relating the asymmetrical intensity distributions observed in the EBSD patterns acquired experimentally and comparing the same with the dynamical electron diffraction simulations. Additionally mean angular error maps were also plotted using automated cross-correlation based approaches to image APDs. Samples grown on substrates with a 4° offcut from the [110] do not show any APDs, whereas samples grown on the exactly oriented substrates contain APDs. The procedures described in our work can be adopted for characterising a wide range of other material systems possessing non-centrosymmetric point groups.