Development of differential sublaminar feedforward inhibitory circuits in CA1 hippocampus requires Satb2.

Pyramidal cells (PCs) in CA1 hippocampus can be classified by their radial position as deep or superficial and organize into subtype-specific circuits necessary for differential information processing. Specifically, superficial PCs receive fewer inhibitory synapses from parvalbumin (PV)-expressing interneurons than deep PCs, resulting in weaker feedforward inhibition of input from CA3 Schaffer collaterals. Using mice, we investigated mechanisms underlying PC differentiation and the development of this inhibitory circuit motif. We found that expression of the transcriptional regulator SATB2 is biased towards superficial PCs during early postnatal development and necessary to suppress PV+ interneuron synapse formation. In the absence of SATB2, the number of PV+ interneuron synaptic puncta surrounding superficial PCs increases during development to match deep PCs. This results in equivalent inhibitory current strength observed in paired whole-cell recordings, and equivalent feedforward inhibition of Schaffer collateral input. Thus, SATB2 is necessary for superficial PC differentiation and biased feedforward inhibition in CA1.

Contemporary diagnostic visual and automated polysomnographic REM sleep without atonia thresholds in isolated REM sleep behavior disorder.

STUDY OBJECTIVES: Accurate diagnosis of isolated rapid eye movement (REM) sleep behavior disorder (iRBD) is crucial due to its injury potential and neurological prognosis. We aimed to analyze visual and automated REM sleep without atonia (RSWA) diagnostic thresholds applicable in varying clinical presentations in a contemporary cohort of patients with iRBD using submentalis (SM) and individual bilateral flexor digitorum superficialis (FDS) and anterior tibialis electromyography limb recordings during polysomnography. METHODS: We analyzed RSWA in 20 patients with iRBD and 20 age-, REM-, apnea-hypopnea index-matched controls between 2017 and 2022 for phasic burst durations, density of phasic, tonic, and "any" muscle activity (number of 3-second mini-epochs containing phasic or tonic muscle activity divided by the total number of REM sleep 3-second mini-epochs), and automated Ferri REM atonia index (RAI). Group RSWA metrics were comparatively analyzed. Receiver operating characteristic curves determined optimized area under the curve (AUC) and maximized specificity and sensitivity diagnostic iRBD RSWA thresholds. RESULTS: All mean RSWA metrics were higher in patients with iRBD than in controls (P < .05), except for selected anterior tibialis measures. Optimized, maximal specificity AUC diagnostic cutoffs for coprimary outcomes were: SM "any" 6.5%, 14.0% (AUC = 92.5%) and combined SM+FDS "any" 15.1%, 27.4% (AUC = 95.8%), while SM burst durations were 0.72, and 0.72 seconds (AUC 90.2%) and FDS RAI = 0.930, 0.888 (AUC 92.8%). CONCLUSIONS: This study provides evidence for current quantitative RSWA diagnostic thresholds in chin and individual 4 limb muscles applicable in different iRBD clinical settings and confirms the key value of SM or SM+FDS to assure accurate iRBD diagnosis. Evolving iRBD recognition underscores the necessity of continuous assessment with future large, prospective, well-harmonized, multicenter polysomnographic analyses. CITATION: Leclair-Visonneau L, Feemster JC, Bibi N, et al. Contemporary diagnostic visual and automated polysomnographic REM sleep without atonia thresholds in isolated REM sleep behavior disorder. J Clin Sleep Med. 2024;20(2):279-291.

Evaluating an under-mattress sleep monitor compared to a peripheral arterial tonometry home sleep apnea test device in the diagnosis of obstructive sleep apnea.

STUDY OBJECTIVES: To evaluate whether or not the apnea-hypopnea index (AHI) from a peripheral arterial tonometry (PAT) home sleep apnea test (HSAT) is equivalent to the AHI provided by the mean of one, three, or seven nights from the Withings Sleep Analyzer (WSA) under-mattress device. METHODS: We prospectively enrolled patients with suspected OSA in whom a PAT-HSAT was ordered. Eligible patients used the WSA for seven to nine nights. PAT data were scored using the device's intrinsic machine learning algorithms to arrive at the AHI using both 3% and 4% desaturation criteria for hypopnea estimations (PAT3%-AHI and PAT4%-AHI, respectively). These were then compared with the WSA-estimated AHI (WSA-AHI). RESULTS: Of 61 patients enrolled, 35 completed the study with valid PAT and WSA data. Of the 35 completers 16 (46%) had at least moderately severe OSA (PAT3%-AHI ≥ 15). The seven-night mean WSA-AHI was 2.13 (95%CI = - 0.88, 5.14) less than the PAT3%-AHI, but 5.64 (95%CI = 2.54, 8.73) greater than the PAT4%-AHI. The accuracy and area under the receiver operating curve (AUC) using the PAT3%-AHI ≥ 15 were 77% and 0.87 and for PAT4%-AHI ≥ 15 were 77% and 0.85, respectively. The one-, three-, or seven-night WSA-AHI were not equivalent to either the 3% or 4% PAT-AHI (equivalency threshold of ± 2.5 using the two one-sided t-test method). CONCLUSIONS: The WSA derives estimates of the AHI unobtrusively over many nights, which may prove to be a valuable clinical tool. However, the WSA-AHI over- or underestimates the PAT-AHI in clinical use, and the appropriate use of the WSA in clinical practice will require further evaluation. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04778748.

Patient values and preferences regarding prognostic counseling in isolated REM sleep behavior disorder.

STUDY OBJECTIVES: Isolated REM sleep behavior disorder (iRBD) carries a high lifetime risk for phenoconversion to a defined neurodegenerative disease (NDD) including Parkinson disease, dementia with Lewy bodies, and multiple system atrophy. We aimed to examine iRBD patient values and preferences regarding prognostic counseling. METHODS: One hundred thirteen iRBD patient participants enrolled in the Mayo Clinic iRBD Patient Registry were sent an email survey concerning their values and preferences concerning NDD prognostic counseling and their experiences following diagnosis with iRBD. RESULTS: Of 81 respondents (71.7% response rate), the majority were men (74.0%) with an average age of 65.7 (±9.7) years. Responses indicated a strong preference toward receiving prognostic information about possible future NDD development. 92.5% of respondents felt knowledge concerning personal NDD risk was important, while 87.6% indicated prognostic discussions were important to maintaining trust in their physician. 95.7% indicated a desire for more information, while only 4.3% desired less information regarding their NDD prognostic risk. Most respondents strongly agreed that prognostic information was important to discuss with their family and friends and inform future life planning, and most expressed interest in learning more about future neuroprotective therapies and symptomatic treatments for parkinsonism and dementia. CONCLUSIONS: Most iRBD patients indicated strong preferences for disclosure of NDD prognostic risk and indicated that prognostic information was important for family discussions and future life planning. Future broader surveys and qualitative studies of clinic-based and ultimately community dwelling iRBD patients' values and preferences are needed to guide appropriately tailored and individualized prognostic counseling approaches following iRBD diagnosis.

Understanding the interaction of proteins to ion exchange chromatographic supports: A surface energetics approach.

Ion exchange chromatography is one of the most widely used chromatographic technique for the separation and purification of important biological molecules. Due to its wide applicability in separation processes, a targeted approach is required to suggest the effective binding conditions during ion exchange chromatography. A surface energetics approach was used to study the interaction of proteins to different types of ion exchange chromatographic beads. The basic parameters used in this approach are derived from the contact angle, streaming potential, and zeta potential values. The interaction of few model proteins to different anionic and cationic exchanger, with different backbone chemistry, that is, agarose and methacrylate, was performed. Generally, under binding conditions, it was observed that proteins having negative surface charges showed strong to lose interaction (20 kT for Hannilase to 0.5 kT for IgG) with different anionic exchangers (having different positive surface charges). On the contrary, anionic exchangers showed almost no interaction (0-0.1 kT) with the positively charged proteins. An inverse behavior was observed for the interaction of proteins to cationic exchangers. The outcome from these theoretical calculations can predict the binding behavior of different proteins under real ion exchange chromatographic conditions. This will ultimately propose a better bioprocess design for protein separation.

Evaluation of the Hypoglycemic Activity of Morchella conica by Targeting Protein Tyrosine Phosphatase 1B.

Morchella conica (M. conica) Pers. is one of six wild edible mushrooms that are widely used by Asian and European countries for their nutritional value. The present study assessed the anti-diabetic potential of M. conica methanolic extract (100 mg/kg body weight) on streptozotocin (STZ)-induced diabetic mice. STZ was used in a single dose of 65 mg/kg to establish diabetic models. Body weights, water/food intake and fasting blood glucose levels were measured. Histopathological analysis of the pancreas and liver were performed to evaluate STZ-induced tissue injuries. In addition, in vitro assays such as α-amylase and protein tyrosine phosphatase 1B (PTP1B) inhibitory, antiglycation, antioxidant and cytotoxicity were performed. The in vitro study indicated potent PTP1B inhibitory potential of M. conica with an IC50 value of 26.5 µg/ml as compared to the positive control, oleanolic acid (IC50 36.2 µg/ml). In vivo investigation showed a gradual decrease in blood sugar level in M. conica-treated mice (132 mg/dl) at a concentration of 100 mg/kg as compared to diabetic mice (346 mg/dl). The extract positively improved liver and kidney damages as were shown by their serum glutamic pyruvic transaminase, serum glutamic oxaloacetate, alkaline phosphatase, serum creatinine and urea levels. Histopathological analysis revealed slight liver and pancreas improvement of mice treated with extract. Cytotoxicity assays displayed lower IC50 values. Based on the present results of the study, it may be inferred that M. conica are rich in bioactive compounds responsible for antidiabetic activity and this mushroom may be a potential source of antidiabetic drug. However, further studies are required in terms of isolation of bioactive compounds to validate the observed results.

Docking Prediction of Levodopa in the Receptor Binding Domain of Spike Protein of SARS-CoV-2.

Levodopa is a prodrug that is converted into dopamine, which replenishes the deficient dopamine in the brain of patients suffering from Parkinsonism. We hypothesize that levodopa may interact with the receptor binding domain of the SARS-CoV-2 and may act as a physical impediment to the viral entry into the host cell.

Distribution and Classification of Dehydrins in Selected Plant Species Using Bioinformatics Approach.

BACKGROUND: Plant growth, reproduction and yields are severely damaged under adverse environmental stresses. These stresses can be either biotic or abiotic, and many stress related proteins are expressed in response to these stresses. Among these proteins dehydrins are reported to have a role primarily in the abiotic stresses. Dehydrins are very diverse proteins and a uniform annotation system is needed for their functional characterization in the future research. OBJECTIVES: The aim of the present work is to identify, classify and analyze the expression of dehydrin proteins under different biotic and abiotic stresses in the selected plant species by using different computational tools. MATERIALS AND METHODS: Prosite database is used for dehydrin proteins identification, and to conform the location of conserved motifs in selected plant species. The dehydrins extracted from uniprot database were annotated, based on the ensemble plant gene id. Subcellular localization was predicted using PSI predictor tool. Dehydrin expression analyses were retrieved form the genevestigator tool. RESULTS: Dehydrins were annotated on the basis of dehydrin gene locus and conserved motifs available in different domain databases. Dehydrins were identified and annotated in Arabidopsis thaliana (13), Glycine max (12), Zea mays (05), Oryza sativa (11), Solanum tuberosum (05), Solanum lycopersicum (06), Triticum aestivum (32) and Vitis vinifera (06). It has been proposed that dehydrins are located primarily in cytosol and nucleus. Based on genevestigater expression analyses the plant species selected for this study contain all the classes of dehydrins, namely YnSKn, Kn, SKn, and YnKn; except class KnS. CONCLUSIONS: Dehydrins are diverse proteins and a uniform classification is introduced for their better characterization. The distribution of dehydrins in different tissues and developmental stages suggest an important function throughout plant growth cycle. It has also been concluded that dehydrins expressed particularly in drought, cold and salt stresses, and may have limited role in heat, anoxia, heavy-metal and biotic stresses as well.

Comparative analysis of the methods used for finding surface energy to investigate protein interaction behavior on chromatographic supports.

Hydrophobic interaction chromatography, an important and effective purification strategy, is generally used for the purification of variety of biomolecules. A basic understanding of the protein interaction behavior is required to effectively separate these biomolecules. A colloidal type extended Derjaguin, Landau, Verwey, and Overbeek calculations were utilized to study the interactions behavior of model proteins to commercially available hydrophobic chromatographic materials that is, Toyopearl Phenyl 650C and Toyopearl Butyl 650C. Physicochemical properties of selected model proteins were achieved by contact angle and zeta potential measurements. The contact angle of chromatographic materials used was achieved through sessile drop method on disrupted beads and capillary penetration method (CPM) on intact beads. The surface properties were further used to calculate the interactions of the proteins to chromatographic supports. The calculated secondary energy minimum of the proteins with the chromatographic materials (from the contact angle values determined through both methods can be correlated with the retention volumes from the real chromatography. The secondary energy minimum values are higher for each protein to the chromatographic materials calculated from the inputs derived through sessile drop method compared to CPM. For instance, immunoglobulin G has secondary energy minimum value of 0.17 kT compared to 0.11 kT, obtained through sessile drop method and CPM, respectively. Average relative values of the energy minimum calculated for all proteins are as 1.51 kT and 1.29 kT for Toyopearl Butyl 650C and Toyopearl Phenyl 650C, respectively, as a conversion factor for estimation of secondary energy minimum for both methods.

Protein adsorption onto monoliths: A surface energetics study.

This part of work was done to explore the basic understanding of the adsorption chromatography by determining the interaction of selected model proteins (n = 5) to monolithic chromatographic materials, with varying densities of butyl and phenyl ligands. Surface energetics approach was applied to study the interaction behavior. The physicochemical properties of the proteins and monolithic chromatographic materials were explored by contact angle and zeta potential values. These values were used to study protein to monolith interaction under various operating conditions. Surface energetics approach allowed the calculation of interaction energy as a function of distance, i.e. energy minimum values. Calculations were performed at various conditions to analyze the effect of major operating parameters on the interaction strength. The interaction strength exposed the hydrophobic nature of the monoliths which increases with increasing ligand density. Further, interaction energy of proteins were higher with monolith with butyl ligand compared to monolith with phenyl ligand. For instance, lactoferrin interaction to monoliths with butyl represents more interaction, i.e. 24.38 kT as compared to monoliths with phenyl i.e. 23.28 kT, keeping lambda as 0.2 nm and salt concentration as 100 mM of ammonium sulphate. Hence, more energy and time will be consumed for elution of proteins immobilized to monoliths with butyl. Similarly, the effect of solid surface for proteins immobilization, effect of ligand density and effect of lambda showed some interesting insights on the interaction behavior. The knowledge generated from the present work will help in the basic understanding as well as development of an efficient, low cost downstream processing design and may mimic the real chromatographic experiments.

A megaporous material harbouring a peptide ligand for affinity IgG purification.

Common limitations of Protein A affinity chromatography include high adsorbent costs, ligand instability and possible ligand leakage. In this study, a short peptide with affinity for IgG was synthesized chemically and subsequently immobilized on a megaporous support. The support was prepared utilising the cryogel technique while the peptide-ligand was covalently immobilised via thiol-epoxy click chemistry. The cryogel support was chemically grafted to increase the number of reaction sites. This adsorbent was designated as "MP-Pep". Adsorption isotherms were employed to evaluate protein binding capacity. A maximum static binding capacity within the range of 30-60 mg/mL was observed for T hIgG. This parameter compares well with other commercial and non-commercial adsorbents, as reported in the literature. As a control material, a Protein A grafted megaporous cryogel was synthesized. Dynamic binding capacity values were obtained by breakthrough analysis. The peptide cryogel showed a dynamic capacity value 9.0 mg/mL in comparison to 9.7 mg/mL in the case of the Protein A based adsorbent. The ratio of dynamic binding capacity to static binding capacity was 20%, indicating suboptimal product capture. However, the advantage of MP-Pep lies in its cost-effective preparations while maintaining a reasonable binding capacity for the targeted product. The presence of cooperative effects during protein binding could also represent an advantage during the processing of a feedstock containing a product in high concentration.

Direct capture of His6-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 µm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 µmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

The role of ligands on protein retention in adsorption chromatography: a surface energetics approach.

Protein adsorption onto hydrophobic chromatographic supports has been investigated using a colloid theory surface energetics approach. The surface properties of commercially available chromatographic beads, Toyopearl Phenyl 650-C, and Toyopearl Butyl 650-C, have been experimentally determined by contact angle and zeta potential measurements. The adsorption characteristics of these beads, which bear the same backbone matrix but harbor different ligands, have been studied toward selected model proteins, in the hydrated as well as dehydrated state. There were two prominent groups of proteins observed with respect to the chromatographic supports presented in this work: loosely retained proteins, which were expected to have lower average interaction energies, and the strongly retained proteins, which were expected to have higher average interaction energies. Results were also compared and contrasted with calculations derived from adsorbent surface energies determined by inverse liquid chromatography. These results showed a good qualitative agreement, and the interaction energy minima obtained from these extended Derjaguin, Landau, Verwey and Overbeek calculations were shown to correlate well with the experimentally determined adsorption behavior of each protein.

Selection of ceramic fluorapatite-binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography.

Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA-specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N-terminal sequence was found in two selected peptides: F4-2 (KPRSMLH) and F5-4 (KPRSVSG). The peptide F5-4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5-4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage-derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins.

Grafted megaporous materials as ion-exchangers for bioproduct adsorption.

Megaporous chromatographic materials were manufactured by a three-step procedure, including backbone synthesis, chemical grafting, and introduction of ion-exchange functionality. The backbone of the adsorbent cylindrical bodies was prepared by polymerization of methacrylic acid and poly(ethylene glycol) diacrylate at sub-zero temperatures. Grafting was performed employing glycidyl methacrylate and a chemical initiator, cerium ammonium nitrate. The degree of grafting was adjusted by modifying the concentration of the initiator in the reaction mixture to a range of values (23, 39, 62, 89, and 105%). Further, the pendant epoxy-groups generated by the previous step were reacted to cation- and anion-exchanging moieties utilizing known chemical routes. Infrared spectroscopy studies confirmed the incorporation of epoxy and ion-exchanger groups to the backbone material. Optimized materials were tested for chromatography applications with model proteins; the dynamic binding capacity, as recorded at 10% breakthrough and 2.0 × 10(-4) m/s superficial velocity, were 350 and 58 mg/g for the cation-exchanger and the anion-exchanger material, respectively. These results may indicate that long tentacle-type polymer brushes were formed during grafting therefore increasing the ability of the megaporous body to efficiently capture macromolecules.

Synthesis and performance of megaporous immobilized metal-ion affinity cryogels for recombinant protein capture and purification.

Megaporous cryogels with metal-ion affinity functionality, which possess enhanced protein-binding ability, were synthesized and their properties were investigated. These highly porous materials (pore sizes up to 100 µm) allowed the direct capture of a recombinant His(6)-tagged protein from a partially clarified extract. The total ligand density of the material was found to be 770 µmol/g. Application of a partially clarified cell extract in order to recover a His(6)-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) yielded 12 mg of highly purified recombinant product per gram of adsorbent. Increased dynamic binding capacities were observed upon larger degrees of grafting, although some reduction in the quality of the system hydrodynamics was also observed. Nevertheless, these immobilized metal-ion affinity cryogels show potential for a convenient single-step purification of recombinant proteins from raw cell extracts without the need for laborious pre-chromatographic sample clean-up procedures.

Extended DLVO calculations expose the role of the structural nature of the adsorbent beads during chromatography.

Protein adsorption onto hydrophobic interaction chromatography supports was studied by a surface-thermodynamics approach. To gather relevant experimental information, contact angle measurements and zeta potential determinations were performed on three different commercial adsorbent beads, Phenyl Sepharose 6 Fast Flow, Toyopearl Phenyl 650-C and Source 15 Phenyl, having soft to rigid backbone structure. Similar information was obtained for a collection of model proteins, lysozyme, bovine serum albumin (BSA), polygalacturonase, aminopeptidase, chymosin, aspartic protease, beta-galactosidase, human immunoglobulin G, and lactoferrin, were evaluated in the hydrated and in the dehydrated state. Based on the mentioned experimental data, calculations were performed to obtain the (interfacial) energy versus distance profiles of nine individual (model) proteins on (commercial) beads of three different types. All of these beads harbored the phenyl-ligand onto a matrix of differentiated chemical nature. Extended Derjaguin, Landau, Verwey, and Overbeek (DLVO) calculations were correlated with actual chromatographic behavior. Typical chromatography conditions were employed. The population of model proteins utilized in this study could be segregated into two groups, according to the minimum values observed for the resulting interaction energy pockets and the corresponding retention volumes (or times) during chromatography. Moreover, trends were also identified as a function of the type of adsorbent bead under consideration. This has revealed the influence of the physicochemical nature of the bead structure on the adsorption process and consequently, on the expected separation behavior.

Synthesis and performance of 3D-megaporous structures for enzyme immobilization and protein capture.

The preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross-linker agent. Ion-exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation-induced grafting. The total ionic capacity of such systems was 1.5 mmol H(+)/g, 1.4 mmol H(+)/g, and 17 mmol H(+)/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ≅7.2 mg bovine serum albumin/g, ≅7.4 hen egg-white lysozyme (HEWL) mg/g, and ≅108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow-through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D-megaporous materials described in this work.