Finding Pattern in the Noise: Persistent Implicit Statistical Knowledge Impacts the Processing of Unpredictable Stimuli.

Humans can extract statistical regularities of the environment to predict upcoming events. Previous research recognized that implicitly acquired statistical knowledge remained persistent and continued to influence behavior even when the regularities were no longer present in the environment. Here, in an fMRI experiment, we investigated how the persistence of statistical knowledge is represented in the brain. Participants (n = 32) completed a visual, four-choice, RT task consisting of statistical regularities. Two types of blocks constantly alternated with one another throughout the task: predictable statistical regularities in one block type and unpredictable ones in the other. Participants were unaware of the statistical regularities and their changing distribution across the blocks. Yet, they acquired the statistical regularities and showed significant statistical knowledge at the behavioral level not only in the predictable blocks but also in the unpredictable ones, albeit to a smaller extent. Brain activity in a range of cortical and subcortical areas, including early visual cortex, the insula, the right inferior frontal gyrus, and the right globus pallidus/putamen contributed to the acquisition of statistical regularities. The right insula, inferior frontal gyrus, and hippocampus as well as the bilateral angular gyrus seemed to play a role in maintaining this statistical knowledge. The results altogether suggest that statistical knowledge could be exploited in a relevant, predictable context as well as transmitted to and retrieved in an irrelevant context without a predictable structure.

G Protein Coupled Receptor 87 (GPR87) in End Stage Kidney and Associated Tumors.

BACKGROUND/AIM: End-stage renal disease (ESRD) and acquired cystic renal disease (ACRD) are characterized by progressive inflammation, structural remodeling and by development of unique cancer types. Eosinophilic-vacuolated and chromophobe-like renal cell carcinoma develop exclusively in ACRD kidney. The aim of the study was to investigate the molecular mechanism of ESRD/ACRD carcinogenesis. MATERIALS AND METHODS: Our previous Affymetrix array analysis detected GPR87 as one of the highly and specifically expressed genes in ESRD/ACRD kidneys. In this study we analyzed normal and ESRD/ACRD kidneys and related tumors for GPR87 expression by PCR, RT-PCR, and immunohistochemistry. RESULTS: Immunohistochemistry revealed a strong GPR87 expression in proliferating epithelial cells in ESRD/ACRD kidneys and in cells of eosinophilic-vacuolated and chromophobe-like renal cell carcinoma. CONCLUSION: GPR87 signaling plays an important role in the structural remodeling of ESRD/ACRD kidney and development of ACRD-associated tumors with unique histology.

Unidirectional hybridization between American paddlefish Polyodon spathula (Walbaum, 1792) and sterlet Acipenser ruthenus (Linnaeus, 1758).

Interspecific hybridizations among sturgeon species are feasible and often bidirectional. The American paddlefish (Polyodon spathula) from Family Polyodontidae and sturgeon species from Family Acipenseridae were reported capable of hybridization, but viable hybrids have been described only in crosses with the American paddlefish as paternal parents. In the reciprocal cross, the hybrids were not viable however embryos start to develop and reach late gastrula and early neurula stages. The goal of this study was to examine the hybridization between the sterlet sturgeon (Acipenser ruthenus) and the American paddlefish. Hybrid and purebred crosses were produced by artificial fertilization. Viable hybrid offspring were harvested (three month old) and verified in the families produced by female sterlet crossing with male American paddlefish. In the reciprocal hybrid crosses with female American paddlefish and male sterlet, the embryos development did not pass over 120 h post fertilization, indicating the unidirectional hybridization between American paddlefish and sterlet. Chromosome counting showed for the same ploidy level of viable hybrid and parent species. Analysis of three microsatellite markers confirmed the unidirectional hybridization between the American paddlefish and the sterlet species. Overall, the inferred genetic cause suggests that unidirectional hybridization between American paddlefish and sterlet may be the case not only for these two species but likely also between American paddlefish and other sturgeon species.

Impact of cellular morphology and three-tiered nuclear grade on progression of conventional renal cell carcinoma.

AIMS AND METHODS: The aims of this study were to evaluate the prognostic impact of cytomorphology and three-tiered grading on tumour-free survival of patients with conventional renal cell carcinoma (cRCC). Formalin-fixed, paraffin-embedded samples from 710 patients were assessed and the results were evaluated according to the clinical data. RESULTS: Kaplan-Meier regression model showed that 90.9% of patients with clear cell, and 50.9% with pure eosinophilic cRCC were free of metastasis during follow-up. The three-triered grading showed a good correlation with progression as 95.2% of patients with of G1 tumours, 66.1% with G2 tumours and only 25.3% with G3 tumours were tumour free (p<0.001). The grading was correlated with cytomorphology and coagulation necrosis. In multivariate analysis, tumour grade and stage were independent prognostic markers (p<0.001). CONCLUSIONS: The three-tiered grading predicts the progression of cRCC irrespectively of cytomorphology. However, the cytomorphology and necrosis show a good correlation with three-tiered grading in estimate disease progression.

Your place or mine? The neural dynamics of personally familiar scene recognition suggests category independent familiarity encoding.

Recognizing a stimulus as familiar is an important capacity in our everyday life. Recent investigation of visual processes has led to important insights into the nature of the neural representations of familiarity for human faces. Still, little is known about how familiarity affects the neural dynamics of non-face stimulus processing. Here we report the results of an EEG study, examining the representational dynamics of personally familiar scenes. Participants viewed highly variable images of their own apartments and unfamiliar ones, as well as personally familiar and unfamiliar faces. Multivariate pattern analyses were used to examine the time course of differential processing of familiar and unfamiliar stimuli. Time-resolved classification revealed that familiarity is decodable from the EEG data similarly for scenes and faces. The temporal dynamics showed delayed onsets and peaks for scenes as compared to faces. Familiarity information, starting at 200 ms, generalized across stimulus categories and led to a robust familiarity effect. In addition, familiarity enhanced category representations in early (250-300 ms) and later (>400 ms) processing stages. Our results extend previous face familiarity results to another stimulus category and suggest that familiarity as a construct can be understood as a general, stimulus-independent processing step during recognition.

Stimulus expectations do not modulate visual event-related potentials in probabilistic cueing designs.

Humans and other animals can learn and exploit repeating patterns that occur within their environments. These learned patterns can be used to form expectations about future sensory events. Several influential predictive coding models have been proposed to explain how learned expectations influence the activity of stimulus-selective neurons in the visual system. These models specify reductions in neural response measures when expectations are fulfilled (termed expectation suppression) and increases following surprising sensory events. However, there is currently scant evidence for expectation suppression in the visual system when confounding factors are taken into account. Effects of surprise have been observed in blood oxygen level dependent (BOLD) signals, but not when using electrophysiological measures. To provide a strong test for expectation suppression and surprise effects we performed a predictive cueing experiment while recording electroencephalographic (EEG) data. Participants (n=48) learned cue-face associations during a training session and were then exposed to these cue-face pairs in a subsequent experiment. Using univariate analyses of face-evoked event-related potentials (ERPs) we did not observe any differences across expected (90% probability), neutral (50%) and surprising (10%) face conditions. Across these comparisons, Bayes factors consistently favoured the null hypothesis throughout the time-course of the stimulus-evoked response. When using multivariate pattern analysis we did not observe above-chance classification of expected and surprising face-evoked ERPs. By contrast, we found robust within- and across-trial stimulus repetition effects. Our findings do not support predictive coding-based accounts that specify reduced prediction error signalling when perceptual expectations are fulfilled. They instead highlight the utility of other types of predictive processing models that describe expectation-related phenomena in the visual system without recourse to prediction error signalling.

End-stage kidney disease: a never healing wound leading to another never healing wound, renal cancer.

BACKGROUND: End-stage kidney disease and acquired cystic kidney disease are the final stages of chronic kidney disease, leading to loss of kidney function and frequent development of tumours. It has been suggested that an inflammatory microenvironment may be responsible for the progressive kidney remodelling and cancer development. METHODS: Our aim was to analyse gene expression suggested to be involved in the remodelling of kidneys in end-stage kidney disease, and in the development of preneoplastic lesions and tumours. Immunohistochemistry was employed to assess the cellular localisation of different genes involved in these pathways on representative tissue sections. RESULTS: Cellular (αSMA positive naïve activated fibroblasts, endothelial cells, macrophages) and non-cellular components (cytokines IL6, TGFß, IL1ß, CSF2, fibronectin, laminin, and matrix modifier proteases MMP9 and MMP12) of the inflammatory microenvironment were expressed in the kidneys of patients with end-stage kidney disease. IL6 and FN1 expressing naïve activated fibroblasts and recruited inflammatory cells were the most abundant cellular components of the inflammatory microenvironment. CONCLUSION: The progressive inflammatory and fibrotic processes in end-stage kidney disease have features recalling those of  a never healing wound and may explain the frequent development of kidney cancer.

The effect of context congruency on fMRI repetition suppression for objects.

The recognition of objects is strongly facilitated when they are presented in the context of other objects (Biederman, 1972). Such contexts facilitate perception and induce expectations of context-congruent objects (Trapp and Bar, 2015). The neural mechanisms underlying these facilitatory effects of context on object processing, however, are not yet fully understood. In the present study, we investigate how context-induced expectations affect subsequent object processing. We used functional magnetic resonance imaging and measured repetition suppression as a proxy for prediction error processing. Participants viewed pairs of alternating or repeated object images which were preceded by context-congruent, context-incongruent or neutral cues. We found a stronger repetition suppression in congruent as compared to incongruent or neutral cues in the object sensitive lateral occipital cortex. Interestingly, this stronger effect was driven by enhanced responses to alternating stimulus pairs in the congruent contexts, rather than by suppressed responses to repeated stimulus pairs, which emphasizes the contribution of surprise-related response enhancement for the context modulation on RS when expectations are violated. In addition, in the congruent condition, we discovered significant functional connectivity between object-responsive and frontal cortical regions, as well as between object-responsive regions and the fusiform gyrus. Our findings indicate that prediction errors, reflected in enhanced brain responses to violated contextual expectations, underlie the facilitating effect of context during object perception.

Novel concept of Wilms' tumor development: involvement of pluripotential cells of ureteric bud.

It is acknowledged that nephron develops after bilateral induction of the metanephric mesenchyma and branching ureteric bud (UB), and that nephrogenic rest and Wilms' tumor (nephroblastoma) arises from impaired differentiation of metanephric blastema. The aim of this study was to obtain more information on the involvement of UB derivatives in nephrogenic rest and Wilms' tumor. We applied immunohistochemistry to analyze nephrogenic rests and Wilms' tumors with mixed histology, including regressive and blastemal types. We used antibodies recognizing UB tip cells (ROBO1, SLIT2, RET), principal cells (AQP2), α- and ß-intercalated cells (SLC26A4, SLC4A1, ATP6V1B1, ATP6V0D2), and their precursors (CA2). Tubules surrounded by tumorous blastemal cells resembling UB tip were positive for RET, ROBO1, and SLIT2 in Wilms' tumor. Moreover, CA2-positive tubular structures and ATP6V1B1- and ATP6V0D2-positive immature non-α- and non-ß-intercalated cells were detected in nephrogenic rest and Wilms' tumor. We suggest that Wilms' tumor is more than nephroblastoma and propose a definition that Wilms tumor is a malignant embryonal neoplasm derived from pluripotential cells of nephrogenic blastema and of ureteric bud tip.

The neural dynamics of familiarity-dependent face identity representation.

Recognizing a face as belonging to a given identity is essential in our everyday life. Clearly, the correct identification of a face is only possible for familiar people, but 'familiarity' covers a wide range-from people we see every day to those we barely know. Although several studies have shown that the processing of familiar and unfamiliar faces is substantially different, little is known about how the degree of familiarity affects the neural dynamics of face identity processing. Here, we report the results of a multivariate EEG analysis, examining the representational dynamics of face identity across several familiarity levels. Participants viewed highly variable face images of 20 identities, including the participants' own face, personally familiar (PF), celebrity and unfamiliar faces. Linear discriminant classifiers were trained and tested on EEG patterns to discriminate pairs of identities of the same familiarity level. Time-resolved classification revealed that the neural representations of identity discrimination emerge around 100 ms post-stimulus onset, relatively independently of familiarity level. In contrast, identity decoding between 200 and 400 ms is determined to a large extent by familiarity: it can be recovered with higher accuracy and for a longer duration in the case of more familiar faces. In addition, we found no increased discriminability for faces of PF persons compared to those of highly familiar celebrities. One's own face benefits from processing advantages only in a relatively late time-window. Our findings provide new insights into how the brain represents face identity with various degrees of familiarity and show that the degree of familiarity modulates the available identity-specific information at a relatively early time window.

FAPα and αSMA mark different subsets of fibroblasts in normal kidney and conventional renal cell carcinoma.

Several studies suggested a correlation between cancer associated fibroblasts (CAF) and cancer progression, but data on conventional renal cell carcinoma (cRCC) is still lacking. We aimed to analyse the impact of αSMA positive myo-CAF and FAPα expressing i-CAF on postoperative relapse of cRCC. We applied immunohistochemistry on tissue-multiarray (TMA) containing 736 consecutively operated cRCC without metastasis at the time of diagnosis. We analysed the correlation between the amount and pattern of αSMA and FAPα expressing CAFs and tumour cells and postoperative tumour relapse. Stromal fibroblasts of each cRCC displayed αSMA immunreaction but only 142 of the 736 tumours showed positive FAPα staining. There was no correlation between the amount of αSMA and or FAPα positive CAFs and tumour progression. However, tumours with large tourtous vessels with strong αSMA positive immunreaction have more then two times higher risk of postoperative tumour relapse (RR=2.198, p = 0.005). Patients with cRCC (57) showing cytoplasmic αSMA staining of tumour cells had a nearly two times higher risk for postoperative progression (RR=1.776, p = 0.014). There is no significant correlation between the density of αSMA or FAPα positive CAFs and postoperative relapse of cRCCs, therefore CAFs in cRCC are not suitable targets for therapy. Further limitation of anti-CAF therapy of cRCC that stromal cells of normal kidney are positive with αSMA antibody.

No semantic information is necessary to evoke general neural signatures of face familiarity: evidence from cross-experiment classification.

Recent theories on the neural correlates of face identification stressed the importance of the available identity-specific semantic and affective information. However, whether such information is essential for the emergence of neural signal of familiarity has not yet been studied in detail. Here, we explored the shared representation of face familiarity between perceptually and personally familiarized identities. We applied a cross-experiment multivariate pattern classification analysis (MVPA), to test if EEG patterns for passive viewing of personally familiar and unfamiliar faces are useful in decoding familiarity in a matching task where familiarity was attained thorough a short perceptual task. Importantly, no additional semantic, contextual, or affective information was provided for the familiarized identities during perceptual familiarization. Although the two datasets originate from different sets of participants who were engaged in two different tasks, familiarity was still decodable in the sorted, same-identity matching trials. This finding indicates that the visual processing of the faces of personally familiar and purely perceptually familiarized identities involve similar mechanisms, leading to cross-classifiable neural patterns.

Context and prediction matter for the interpretation of social interactions across species.

Predictions about others' future actions are crucial during social interactions, in order to react optimally. Another way to assess such interactions is to define the social context of the situations explicitly and categorize them according to their affective content. Here we investigate how humans assess aggressive, playful and neutral interactions between members of three species: human children, dogs and macaques. We presented human participants with short video clips of real-life interactions of dyads of the three species and asked them either to categorize the context of the situation or to predict the outcome of the observed interaction. Participants performed above chance level in assessing social situations in humans, in dogs and in monkeys. How accurately participants predicted and categorized the situations depended both on the species and on the context. Contrary to our hypothesis, participants were not better at assessing aggressive situations than playful or neutral situations. Importantly, participants performed particularly poorly when assessing aggressive behaviour for dogs. Also, participants were not better at assessing social interactions of humans compared to those of other species. We discuss what mechanism humans use to assess social situations and to what extent this skill can also be found in other social species.

Optimizing the gonadoliberin dosage and evaluating the egg quality in the preseason and seasonal artificial reproduction of pond-reared sterlet Acipenser ruthenus.

Sterlet Acipenser ruthenus is the smallest in size, early maturing, yet third in production scale among the sturgeon species and as such presents a good model to study reproductive biology. The present study aimed to evaluate the effects of preseason artificial reproduction and reduction of hormonal dosage on the egg quality in pond-reared sterlet. Therefore, a series of three trials were conducted in the period 2021 and 2022 (21 and 22) that evaluated three dosages of mammalian gonadotropin releasing hormone analog des-Gly10-(d-Ala6)-GnRH (40, 20, and 10 µg/kg) in both preseason (January) and seasonal (April) spawning batches (PRE and SEA). The greatest embryo survival was achieved in SEA-21-10 (84.8 ± 5.8 %) and the lowest in PRE-21-40 (31.0 ± 40.7 %). Overall results indicated that a dosage of 10 µg/kg leads to the highest embryo survival. Considering the season, latency time was significantly longer in preseason reproduction being on average about 6 h prolonged (35.1 ± 2.8 h vs. 41.4 ± 2.9 h) and it directly affected the embryo survival (linear correlation r = - 0.769, P = 0.003). Likewise, preseason reproduction yields more variable egg quality, due to impaired performance of later ovulating fish most likely due to lower oocyte maturation competence. The present study found expected latency time at 14 °C to be 37-41 h in the preseason and 31-41 h in season, while the recommended dosage is 10 µg/kg des-Gly10-(d-Ala6)-GnRH. Future studies should develop in vitro tools to evaluate the oocyte maturational competence to improve the egg quality in the preseason.

The effect of short-term training on repetition probability effects for non-face objects.

The magnitude of repetition suppression (RS) is modulated by the probability of stimulus repetitions when measured by fMRI. This repetition probability effect (P(rep)) is usually expressed in a stronger RS when the probability of repetition trials is higher when compared to blocks with less frequent repetitions. Previous studies have suggested that the P(rep) effect depends on the extensive long-term experience of participants with the stimuli. At the same time, the how short-term learning affects RS and its modulation by P(rep) remains largely unknown. To address this issue, we used fMRI and measured the RS and its modulation by P(rep) for non-face objects (cars) before and after a 10-day long perceptual learning (PL) period and for unfamiliar faces as control. The results showed a significant P(rep) effect for faces within the Fusiform Face Area (FFA) and for cars within the Lateral Occipital Complex (LO) in the pre-training fMRI measurement session. Following the PL period, participants exhibited strong improvements in the subordinate categorization of the trained stimuli. Surprisingly, the magnitude of RS did not change as a function of training, but the P(rep) effect was absent in the post-training fMRI sessions for both stimulus categories. These results suggest that the predictive processes, measured by P(rep) modulation of RS, may be modulated by the short-term perceptual learning experience.

Atypical prosopagnosia following right hemispheric stroke: A 23-year follow-up study with M.T.

Most findings on prosopagnosia to date suggest preserved voice recognition in prosopagnosia (except in cases with bilateral lesions). Here we report a follow-up examination on M.T., suffering from acquired prosopagnosia following a large unilateral right-hemispheric lesion in frontal, parietal, and anterior temporal areas excluding core ventral occipitotemporal face areas. Twenty-three years after initial testing we reassessed face and object recognition skills [Henke, K., Schweinberger, S. R., Grigo, A., Klos, T., & Sommer, W. (1998). Specificity of face recognition: Recognition of exemplars of non-face objects in prosopagnosia. Cortex, 34(2), 289-296]; [Schweinberger, S. R., Klos, T., & Sommer, W. (1995). Covert face recognition in prosopagnosia - A dissociable function? Cortex, 31(3), 517-529] and additionally studied voice recognition. Confirming the persistence of deficits, M.T. exhibited substantial impairments in famous face recognition and memory for learned faces, but preserved face matching and object recognition skills. Critically, he showed substantially impaired voice recognition skills. These findings are congruent with the ideas that (i) prosopagnosia after right anterior temporal lesions can persist over long periods > 20 years, and that (ii) such lesions can be associated with both facial and vocal deficits in person recognition.

Characterizing the shared signals of face familiarity: Long-term acquaintance, voluntary control, and concealed knowledge.

In a recent study using cross-experiment multivariate classification of EEG patterns, we found evidence for a shared familiarity signal for faces, patterns of neural activity that successfully separate trials for familiar and unfamiliar faces across participants and modes of familiarization. Here, our aim was to expand upon this research to further characterize the spatio-temporal properties of this signal. By utilizing the information content present for incidental exposure to personally familiar and unfamiliar faces, we tested how the information content in the neural signal unfolds over time under different task demands - giving truthful or deceptive responses to photographs of genuinely familiar and unfamiliar individuals. For this goal, we re-analyzed data from two previously published experiments using within-experiment leave-one-subject-out and cross-experiment classification of face familiarity. We observed that the general face familiarity signal, consistent with its previously described spatio-temporal properties, is present for long-term personally familiar faces under passive viewing, as well as for acknowledged and concealed familiarity responses. Also, central-posterior regions contain information related to deception. We propose that signals in the 200-400 ms window are modulated by top-down task-related anticipation, while the patterns in the 400-600 ms window are influenced by conscious effort to deceive. To our knowledge, this is the first report describing the representational dynamics of concealed knowledge for faces, using time-resolved multivariate classification.

A neural measure of the degree of face familiarity.

Recognizing a face as familiar is essential in our everyday life. However, 'familiarity' covers a wide range - from people we see every day to those we barely know. Although face recognition is studied extensively, little is known about how the degree of familiarity affects neural face processing, despite the critical social importance of this dimension. Here we report the results of a multivariate cross-classification EEG experiment, where we study the temporal representational dynamics of the degree of familiarity. Participants viewed highly variable face images of 20 identities. Importantly, we measured face familiarity using subjective familiarity ratings in addition to testing explicit knowledge and reaction times in a face matching task. A machine learning algorithm, trained to discriminate familiar and unfamiliar faces from a separate study, was used to predict the degree of face familiarity from the pattern of the EEG data. We found that the neural representations of the degree of familiarity emerge between 400 - 600 msec post-stimulus onset for famous persons. The correlation between decoding performance and behavioral familiarity was more reliable, occurred earlier and lasted longer when personally familiar and viewers' own faces were included in the analysis. Our findings provide new insights into how the brain represents faces with various degrees of familiarity and show that the degree of familiarity can be decoded reliably from the EEG at a relatively late time window. These results support the idea that representations of familiar faces form part of a general neural signature of the familiarity component of recognition memory processes.

Repetition probability effects for Chinese characters and German words in the visual word form area.

The magnitude of repetition suppression (RS), measured by fMRI, is modulated by the probability of repetitions (P(rep)) for various sensory stimulus categories. It has been suggested that for visually presented simple letters this P(rep) effect depends on the prior practices of the participants with the stimuli. Here we tested further if previous experiences affect the neural mechanisms of RS, leading to the modulatory effects of stimulus P(rep), for more complex lexical stimuli as well. We measured the BOLD signal in the Visual Word Form Area (VWFA) of native Chinese and German participants and estimated the P(rep) effects for Chinese characters and German words. The results showed a significant P(rep) effect for stimuli of the mother tongue in both participant groups. Interestingly, Chinese participants, learning German as a second language, also showed a significant P(rep) modulation of RS for German words while the German participants who had no prior experiences with the Chinese characters showed no such effects. Our findings suggest that P(rep) effects on RS are manifest for visual word processing as well, but only for words of a language with which participants are highly familiar. These results support further the idea that predictive processes, estimated by P(rep) modulations of RS, require prior experiences.

Fish with larger pre-seasonal oocytes yields lower egg quality in season - A case study of outdoor-cultured domesticated Pikeperch (Sander lucioperca).

Seasonal reproduction of domesticated pikeperch has been the most critical spawning batch in several European countries. The present study aimed to monitor oocyte growth between mid-November and seasonal spawning to evaluate if oocyte growth trends may predict egg quality. Nineteen sexually mature females were monitored for oocyte sizes every two months. In mid-March, fish were transported to the indoor facility and artificially reproduced. Sixteen females ovulated and egg quality parameters were assessed and further related to oocyte sizes measured in November, January, and March, as well as to size increments between samplings. Based on the oocyte diameters, fish were assigned to two size groups, and the egg quality was compared. Oocyte growth was greater between the first two samplings compared to the consecutive period (150.1 ± 16.5 µm vs 24.7 ± 20.4 µm). Diameters assessed in November and March positively correlated with oil globule fragmentation, while diameters assessed in November and January positively correlated with larval malformation. Although in January larger oocytes showed better embryo survival than smaller ones (60.6 ± 9.5% vs 37.8 ± 23.2%,) they were characterized with greater percent of malformed larvae (25.0 ± 22.0% vs 5.4 ± 3.9%). It appears that fish with bigger oocytes in pre-season have an affinity toward increased embryo survival, however, they seem to have an issue with oil globule fragmentation and larvae malformation in case of seasonal spawning. Therefore, the selection of breeders for either pre-seasonal or seasonal reproduction batch according to the November oocyte size is recommended.