The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

• Klíčová slova
• bottom-up proteomics, method development, peptide properties, peptide separation, protein separation, reversed-phase liquid chromatography, tutorial,
• MeSH
• chromatografie s reverzní fází * metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• peptidy analýza   MeSH
• proteomika * metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• peptidy MeSH

Trypsin dominates bottom-up proteomics, but there are reasons to consider alternative enzymes. Improving sequence coverage, exposing proteomic "dark matter," and clustering post-translational modifications in different ways and with higher-order drive the pursuit of reagents complementary to trypsin. Additionally, enzymes that are easy to use and generate larger peptides that capitalize upon newer fragmentation technologies should have a place in proteomics. We expressed and characterized recombinant neprosin, a novel prolyl endoprotease of the DUF239 family, which preferentially cleaves C-terminal to proline residues under highly acidic conditions. Cleavage also occurs C-terminal to alanine with some frequency, but with an intriguingly high "skipping rate." Digestion proceeds to a stable end point, resulting in an average peptide mass of 2521 units and a higher dependence upon electron-transfer dissociation for peptide-spectrum matches. In contrast to most proline-cleaving enzymes, neprosin effectively degrades proteins of any size. For 1251 HeLa cell proteins identified in common using trypsin, Lys-C, and neprosin, almost 50% of the neprosin sequence contribution is unique. The high average peptide mass coupled with cleavage at residues not usually modified provide new opportunities for profiling clusters of post-translational modifications. We show that neprosin is a useful reagent for reading epigenetic marks on histones. It generates peptide 1-38 of histone H3 and peptide 1-32 of histone H4 in a single digest, permitting the analysis of co-occurring post-translational modifications in these important N-terminal tails.

• MeSH
• HeLa buňky MeSH
• histony chemie   metabolismus   MeSH
• lidé MeSH
• peptidy metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• proteasy metabolismus   MeSH
• proteomika metody   MeSH
• rekombinantní proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histony MeSH
• peptidy MeSH
• proteasy MeSH
• rekombinantní proteiny MeSH

Schizophrenia (SCHZ) notably impacts various human perceptual modalities, including vision. Prior research has identified marked abnormalities in perceptual organization in SCHZ, predominantly attributed to deficits in bottom-up processing. Our study introduces a novel paradigm to differentiate the roles of top-down and bottom-up processes in visual perception in SCHZ. We analysed eye-tracking fixation ground truth maps from 28 SCHZ patients and 25 healthy controls (HC), comparing these with two mathematical models of visual saliency: one bottom-up, based on the physical attributes of images, and the other top-down, incorporating machine learning. While the bottom-up (GBVS) model revealed no significant overall differences between groups (beta = 0.01, p = 0.281, with a marginal increase in SCHZ patients), it did show enhanced performance by SCHZ patients with highly salient images. Conversely, the top-down (EML-Net) model indicated no general group difference (beta = -0.03, p = 0.206, lower in SCHZ patients) but highlighted significantly reduced performance in SCHZ patients for images depicting social interactions (beta = -0.06, p < 0.001). Over time, the disparity between the groups diminished for both models. The previously reported bottom-up bias in SCHZ patients was apparent only during the initial stages of visual exploration and corresponded with progressively shorter fixation durations in this group. Our research proposes an innovative approach to understanding early visual information processing in SCHZ patients, shedding light on the interplay between bottom-up perception and top-down cognition.

• Publikační typ
• časopisecké články MeSH

Visual perception is one of the basic tools for exploring the world. However, in schizophrenia, this modality is disrupted. So far, there has been no clear answer as to whether the disruption occurs primarily within the brain or in the precortical areas of visual perception (the retina, visual pathways, and lateral geniculate nucleus [LGN]). A web-based comprehensive search of peer-reviewed journals was conducted based on various keyword combinations including schizophrenia, saliency, visual cognition, visual pathways, retina, and LGN. Articles were chosen with respect to topic relevance. Searched databases included Google Scholar, PubMed, and Web of Science. This review describes the precortical circuit and the key changes in biochemistry and pathophysiology that affect the creation and characteristics of the retinal signal as well as its subsequent modulation and processing in other parts of this circuit. Changes in the characteristics of the signal and the misinterpretation of visual stimuli associated with them may, as a result, contribute to the development of schizophrenic disease.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.

• Klíčová slova
• Pig, Sperm capacitation, Sperm proteomics, Ubiquitin-proteasome system,
• MeSH
• kapacitace spermií * fyziologie   MeSH
• prasata MeSH
• proteasomový endopeptidasový komplex * metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika * metody   MeSH
• spermie * metabolismus   fyziologie   MeSH
• ubikvitin * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteasomový endopeptidasový komplex * MeSH
• proteom MeSH
• ubikvitin * MeSH

Some rats [sign-trackers (STs)] are prone to attribute incentive salience to reward cues, which can manifest as a propensity to approach and contact pavlovian cues, and for addiction-like behavior. STs also exhibit poor attentional performance, relative to goal-trackers (GTs), which is associated with attenuated acetylcholine (ACh) levels in prefrontal cortex (Paolone et al., 2013). Here, we demonstrate a cellular mechanism, linked to ACh synthesis, that accounts for attenuated cholinergic capacity in STs. First, we found that electrical stimulation of the basal forebrain increased cortical choline transporter (CHT)-mediated choline transport in GTs, paralleled by a redistribution of CHTs to the synaptic plasma membrane. Neither increases in choline uptake nor translocation of CHTs occurred in STs. Second, and consistent with uptake/translocation alterations, STs demonstrated a reduced ability to support cortical ACh release in vivo compared with GTs after reverse-dialysis to elevate extracellular potassium levels. Third, rats were significantly more likely to develop sign-tracking behavior if treated systemically before pavlovian conditioned approach training with the CHT inhibitor VU6001221. Consistent with its proposed mechanisms, administration of VU6001221 attenuated potassium-evoked ACh levels in prefrontal cortex measured with in vivo microdialysis. We propose that loss of CHT-dependent activation of cortical cholinergic activity in STs degrades top-down executive control over behavior, producing a bias for bottom-up or stimulus-driven attention. Such an attentional bias contributes to nonadaptive reward processing and thus identifies a novel mechanism that can support psychopathology, including addiction.SIGNIFICANCE STATEMENT The vulnerability for addiction-like behavior has been associated with psychological traits, such as the propensity to attribute incentive salience to reward cues that is modeled in rats by sign-tracking behavior. Sign-trackers tend to approach and contact cues associated with reward, whereas their counterparts, the goal-trackers, have a preference for approaching the location of the reward. Here, we show that the capacity of presynaptic cholinergic synapses to respond to stimulation by elevating presynaptic choline uptake and releasing acetylcholine is attenuated in sign-trackers. Furthermore, pharmacological inhibition of choline transport induced sign-tracking behavior. Our findings suggest that reduced levels of cholinergic neuromodulation can mediate an attentional bias toward reward-related cues, thereby allowing such cues to exert relatively greater control over behavior.

• Klíčová slova
• acetylcholine, addiction, attention, choline transporter, motivation, sign-tracking,
• MeSH
• acetylcholin metabolismus   MeSH
• biologické markery metabolismus   MeSH
• cholin metabolismus   MeSH
• kauzalita MeSH
• krysa rodu Rattus MeSH
• membránové transportní proteiny metabolismus   MeSH
• neurony cholinergní fyziologie   MeSH
• neurotransmiterové látky metabolismus   MeSH
• odklon pozornosti fyziologie   MeSH
• odměna * MeSH
• podněty MeSH
• potkani Sprague-Dawley MeSH
• presynaptická zakončení metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• acetylcholin MeSH
• biologické markery MeSH
• cholin MeSH
• choline transporter MeSH Prohlížeč
• membránové transportní proteiny MeSH
• neurotransmiterové látky MeSH

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

• MeSH
• buněčná membrána metabolismus   MeSH
• cytoskelet metabolismus   MeSH
• lipidové dvojvrstvy chemie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• savci metabolismus   MeSH
• septiny * chemie   genetika   metabolismus   MeSH
• unilamelární lipozómy * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipidové dvojvrstvy MeSH
• septiny * MeSH
• unilamelární lipozómy * MeSH

Nanosized drug crystals have been reported with enhanced apparent solubility, bioavailability, and therapeutic efficacy compared to microcrystal materials, which are not suitable for parenteral administration. However, nanocrystal design and development by bottom-up approaches are challenging, especially considering the non-standardized process parameters in the injection step. This work aims to present a systematic step-by-step approach through Quality-by-Design (QbD) and Design of Experiments (DoE) for synthesizing drug nanocrystals by a semi-automated nanoprecipitation method. Curcumin is used as a drug model due to its well-known poor water solubility (0.6 µg mL-1, 25 °C). Formal and informal risk assessment tools allow identifying the critical factors. A fractional factorial 24-1 screening design evaluates their impact on the average size and polydispersity of nanocrystals. The optimization of significant factors is done by a Central Composite Design. This response surface methodology supports the rational design of the nanocrystals, identifying and exploring the design space. The proposed joint approach leads to a reproducible, robust, and stable nanocrystalline preparation of 316 nm with a PdI of 0.217 in compliance with the quality profile. An orthogonal approach for particle size and polydispersity characterization allows discarding the formation of aggregates. Overall, the synergy between advanced data analysis and semi-automated standardized nanocrystallization of drugs is highlighted.

• Klíčová slova
• design space, nanocrystals, orthogonal characterization, response surface methodology, solvent–antisolvent precipitation,
• MeSH
• automatizace MeSH
• krystalizace MeSH
• kurkumin chemie   MeSH
• léčivé přípravky chemie   MeSH
• nanočástice * chemie   MeSH
• velikost částic MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kurkumin MeSH
• léčivé přípravky MeSH

BACKGROUND: The default mode network (DMN) decreases its activity when switching from a resting state to a cognitive task condition, while activity of the network engaged in the given task increases. Visual processing is typically disturbed in Parkinson's disease dementia (PDD). OBJECTIVE: Using functional MRI, we studied the DMN effective connectivity patterns in PDD as compared with cognitively normal patients with Parkinson's disease (PD) and healthy controls (HC) when switching from baseline to a visual cognitive task condition. METHODS: In all, 14 PDD, 18 PD, and 18 age-matched healthy controls participated in this functional MRI study. We used a psychophysiological interaction analysis with the precuneus (PCu) as a seed. The threshold was set at p(FWE) <0.05. RESULTS: The healthy controls showed greater PCu connectivity with the bilateral middle temporal/middle occipital gyri at baseline than during the task condition. The correlation direction changed from positive to negative. Both PD and PDD showed disturbed DMN connectivity with the brain regions that are involved in bottom-up visual processing. In PD, we also found impaired integration of the areas engaged in the ventral attentional network, which might reflect specific attentional deficits observed during the early course of PD. In mild PDD, we detected increased engagement of areas involved in the dorsal attentional network, which corresponds to increased top-down control in this patient group as compared to the healthy controls. CONCLUSION: Our results show impaired dynamic interplay between large scale brain networks in PD that spread far beyond the motor system.

• Klíčová slova
• Default mode network, Parkinson's disease, dementia, functional MRI, precuneus, visual processing,
• MeSH
• atrofie etiologie   patologie   MeSH
• hyperkinetická porucha etiologie   MeSH
• kognitivní poruchy etiologie   patologie   MeSH
• kyslík krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• magnetická rezonanční tomografie MeSH
• mapování mozku MeSH
• modely neurologické MeSH
• nervové dráhy krevní zásobení   patologie   MeSH
• neuropsychologické testy MeSH
• Parkinsonova nemoc komplikace   patologie   MeSH
• počítačové zpracování obrazu MeSH
• poruchy zraku etiologie   patologie   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• studie případů a kontrol MeSH
• světelná stimulace MeSH
• temenní lalok krevní zásobení   patologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyslík MeSH

The objective is to study the involvement of the posterior medial cortex (PMC) in encoding and retrieval by visual and auditory memory processing. Intracerebral recordings were studied in two epilepsy-surgery candidates with depth electrodes implanted in the retrosplenial cingulate, precuneus, cuneus, lingual gyrus and hippocampus. We recorded the event-related potentials (ERP) evoked by visual and auditory memory encoding-retrieval tasks. In the hippocampus, ERP were elicited in the encoding and retrieval phases in the two modalities. In the PMC, ERP were recorded in both the encoding and the retrieval visual tasks; in the auditory modality, they were recorded in the retrieval task, but not in the encoding task. In conclusion, the PMC is modality dependent in memory processing. ERP is elicited by memory retrieval, but it is not elicited by auditory encoding memory processing in the PMC. The PMC appears to be involved not only in higher-order top-down cognitive activities but also in more basic, rather than bottom-up activities.

• MeSH
• akustická stimulace MeSH
• dospělí MeSH
• elektroencefalografie metody   MeSH
• epilepsie patofyziologie   MeSH
• implantované elektrody MeSH
• lidé MeSH
• mozek fyziologie   MeSH
• paměť fyziologie   MeSH
• počítačové zpracování signálu MeSH
• sluchové evokované potenciály fyziologie   MeSH
• světelná stimulace MeSH
• zrakové evokované potenciály fyziologie   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH