Development and Validation of a Proximity Labeling Fusion Protein Construct to Identify the Protein-Protein Interactions of Transcription Factors.

Dynamic interactions between transcription factors govern changes in gene expression that mediate changes in cell state accompanying injury response and regeneration. Transcription factors frequently function as obligate dimers whose activity is often modulated by post-translational modifications. These critical and often transient interactions are not easily detected by traditional methods to investigate protein-protein interactions. This chapter discusses the design and validation of a fusion protein involving a transcription factor tethered to a proximity labeling ligase, APEX2. In this technique, proteins are biotinylated within a small radius of the transcription factor of interest, regardless of time of interaction. Here we discuss the validations required to ensure proper functioning of the transcription factor proximity labeling tool and the sample preparation of biotinylated proteins for mass spectrometry analysis of putative protein interactors.

A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium.

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

Montelukast deprescribing in outpatient specialty clinics: A single center cross-sectional study.

Objective: To identify and evaluate montelukast deprescribing in outpatient specialty clinics. Methods: This was a single-center, retrospective, cross-sectional study conducted at an academic health system in the southern US including 21 specialty clinics. Subjects included adults ≥18 years with an active prescription for montelukast who attended at least one appointment in pulmonology, otolaryngology, or neurology outpatient specialty clinics between January 1, 2021 to December 31, 2022. Patients <18 years and those with diagnoses of uncontrolled asthma or allergic rhinitis were excluded. Outcomes assessed included the frequency and period prevalence of montelukast deprescribing, defined by a documented montelukast discontinuation within the medical record, and evaluation of reasoning for discontinuation mentioned in visit notes. Results: There were 1152 patients who met inclusion criteria. Of these, 43 (3.7 %) experienced a montelukast deprescribing event: 18 (41.9 %) in neurology, 13 (30.2 %) in otolaryngology, and 12 (27.9 %) in pulmonology. Documented reasons for deprescribing were only available for 11 patients (25.6 %); reasons for deprescribing included patient-provider shared decision-making regarding the Black Box Warning [n = 5 (11.6 %)], inadequate treatment response [n = 3 (7.0 %)], suicidal thought development [n = 1 (2.3 %)], adverse drug event [n = 1 (2.3 %)], and pregnancy planning [n = 1 (2.3 %)]. Conclusion: Montelukast deprescribing rates were less than 5 % in outpatient specialty clinics. Factors associated with montelukast deprescribing beget further investigation.

Enhancing transjugular intrahepatic portosystemic shunt procedure efficiency with digital subtraction angiography image overlay technology in esophagogastric variceal bleeding.

BACKGROUND: Transjugular intrahepatic portosystemic shunt (TIPS) is a pivotal intervention for managing esophagogastric variceal bleeding in patients with chronic hepatic schistosomiasis. AIM: To evaluate the efficacy of digital subtraction angiography image overlay technology (DIT) in guiding the TIPS procedure. METHODS: We conducted a retrospective analysis of patients who underwent TIPS at our hospital, comparing outcomes between an ultrasound-guided group and a DIT-guided group. Our analysis focused on the duration of the portosystemic shunt puncture, the number of punctures needed, the total surgical time, and various clinical indicators related to the surgery. RESULTS: The study included 52 patients with esophagogastric varices due to chronic hepatic schistosomiasis. Results demonstrated that the DIT-guided group experienced significantly shorter puncture times (P < 0.001) and surgical durations (P = 0.022) compared to the ultrasound-guided group. Additionally, postoperative assessments showed significant reductions in aspartate aminotransferase, B-type natriuretic peptide, and portal vein pressure in both groups. Notably, the DIT-guided group also showed significant reductions in total bilirubin (P = 0.001) and alanine aminotransferase (P = 0.023). CONCLUSION: The use of DIT for guiding TIPS procedures highlights its potential to enhance procedural efficiency and reduce surgical times in the treatment of esophagogastric variceal bleeding in patients with chronic hepatic schistosomiasis.

Cerebral perfusion in patients with unilateral internal carotid artery occlusion by dual post-labeling delays arterial spin labeling imaging.

BACKGROUND: Global and regional cerebral blood flow (CBF) changes in patients with unilateral internal carotid artery occlusion (ICAO) are unclear when the dual post-labeling delays (PLD) arterial spin labeling (ASL) magnetic resonance imaging (MRI) technique is used. Manual delineation of regions of interest for CBF measurement is time-consuming and laborious. AIM: To assess global and regional CBF changes in patients with unilateral ICAO with the ASL-MRI perfusion technique. METHODS: Twenty hospitalized patients with ICAO and sex- and age-matched controls were included in the study. Regional CBF was measured by Dr. Brain's ASL software. The present study evaluated differences in global, middle cerebral artery (MCA) territory, anterior cerebral artery territory, and Alberta Stroke Program Early Computed Tomography Score (ASPECTS) regions (including the caudate nucleus, lentiform nucleus, insula ribbon, internal capsule, and M1-M6) and brain lobes (including frontal, parietal, temporal, and insular lobes) between ICAO patients and controls at PLD 1.5 s and PLD 2.5 s. RESULTS: When comparing CBF between ICAO patients and controls, the global CBF in ICAO patients was lower at both PLD 1.5 s and PLD 2.5 s; the CBF on the occluded side was lower in 15 brain regions at PLD 1.5 s, and it was lower in 9 brain regions at PLD 2.5 s; the CBF in the contralateral hemisphere was lower in the caudate nucleus and internal capsule at PLD 1.5 s and in M6 at PLD 2.5 s. The global CBF in ICAO patients was lower at PLD 1.5 s than at PLD 2.5 s. The ipsilateral CBF at PLD 1.5 s was lower than that at PLD 2.5 s in 15 regions, whereas the contralateral CBF was lower at PLD 1.5 s than at PLD 2.5 s in 12 regions. The ipsilateral CBF was lower than the contralateral CBF in 15 regions at PLD 1.5 s, and in M6 at PLD 2.5 s. CONCLUSION: Unilateral ICAO results in hypoperfusion in the global and MCA territories, especially in the ASPECTS area. Dual PLD settings prove more suitable for accurate CBF quantification in ICAO.

Cerebrospinal fluid flow in small-breed dogs with idiopathic epilepsy observed using time-spatial labeling inversion pulse images: a preliminary study.

Cerebrospinal fluid (CSF) circulation diseases, such as hydrocephalus and syringomyelia, are common in small-breed dogs. In human patients with CSF circulation diseases, time-spatial labeling inversion pulse (time-SLIP) sequence performed to evaluate CSF flow before and after treatment allows visualization of the restoration of CSF movement. However, studies evaluating CSF flow using the time-SLIP method in small-breed dogs are limited. Therefore, the present study aimed to evaluate intracranial CSF flow on time-SLIP images in small-breed dogs with idiopathic epilepsy, as an alternative model to healthy dogs. Time-SLIP images were obtained at two sites: 1) the mesencephalic aqueduct (MA) area (third ventricle, MA, and brain-base subarachnoid space [SAS]) and 2) the craniocervical junction area (fourth ventricle, brainstem, and cervical spinal cord SAS) to allow subsequent evaluation of the rostral and caudal CSF flow using subjective and objective methods. In total, six dogs were included. Caudal flow at the MA and brain-base SAS and rostral flow in the brainstem SAS were subjectively and objectively observed in all and 5/6 dogs, respectively. Objective evaluation revealed that a significantly smaller movement of the CSF, assessed as the absence of CSF flow by subjective evaluation, could be detected in some areas. In small-breed dogs, the MA, brain-base, and brainstem SAS would be appropriate areas for evaluating CSF movement, either in the rostral or caudal flows on time-SLIP images. In areas where CSF movement cannot detected by subjective methods, an objective evaluation should be conducted.

Common SYNE2 Genetic Variant Associated With Atrial Fibrillation Lowers Expression of Nesprin-2α1 With Downstream Effects on Nuclear and Electrophysiological Traits.

BACKGROUND: Atrial fibrillation GWAS (genome-wide association studies) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding Nesprin-2, which connects the nuclear membrane with the cytoskeleton. METHODS: Reporter gene vector transfection and CRISPR-Cas9 editing were used to identify the causal variant regulating the expression of SYNE2α1. After SYNE2 knockdown or SYNE2α1 overexpression in human stem cell-derived cardiomyocytes, nuclear phenotypes were assessed by imaging and atomic force microscopy. Gene expression was assessed by RNAseq and gene set enrichment analysis. Fura-2 AM staining assessed calcium transients. Optical mapping assessed action potential duration and conduction velocity. RESULTS: The risk allele of rs1152591 had lower promoter and enhancer activity and was significantly associated with lower expression of the short SYNE2α1 isoform in human stem cell-derived cardiomyocytes, without an effect on the expression of the full-length SYNE2 mRNA. SYNE2α1 overexpression had dominant negative effects on the nucleus with its overexpression or SYNE2 knockdown leading to increased nuclear area and decreased nuclear stiffness. Gene expression results from SYNE2α1 overexpression demonstrated both concordant and nonconcordant effects with SYNE2 knockdown. SYNE2α1 overexpression had a gain of function on electrophysiology, leading to significantly faster calcium reuptake and decreased assessed action potential duration, while SYNE2 knockdown showed both shortened assessed action potential duration and decreased conduction velocity. CONCLUSIONS: rs1152591 was identified as a causal atrial fibrillation variant, with the risk allele decreasing SYNE2α1 expression. Downstream effects of SYNE2α1 overexpression include changes in nuclear stiffness and electrophysiology, which may contribute to the mechanism for the risk allele's association with AF.

The α3ß4 nAChR tissue distribution identified by fluorescent α-conotoxin [D11A]LvIA.

α3ß4, a vital subtype of neuronal nicotinic acetylcholine receptors (nAChRs), is widely distributed in the brain, ganglia, and adrenal glands, associated with addiction and neurological diseases. However, the lack of specific imaging tools for α3ß4 nAChRs has hindered the investigation of their tissue distribution and functions. [D11A]LvIA, a peptide derived from marine cone snails, demonstrates high affinity and potency for α3ß4 nAChRs, making it a valuable pharmacological tool for studying this receptor subtype. In this study, three fluorescent conjugates of [D11A]LvIA were synthesized using 6-TAMRA-SE (R), Cy3-NHS-ester (Cy3), and BODIPY-FL NHS ester (BDP) dyes. The electrophysiological activities were assessed in Xenopus laevis oocytes by two-electrodes voltage clamp (TEVC). [D11A]LvIA-Cy3 and [D11A]LvIA-BDP show improved selectivity and affinity, with IC50 values of 512.70 nM and 343.50 nM, respectively, and [D11A]LvIA-Cy3 exhibits better stability in cerebrospinal fluid (CSF). Utilizing [D11A]LvIA-Cy3, we successfully visualized the distribution of α3ß4 nAChRs in rat trigeminal ganglia, retina, adrenal glands, and various brain regions. This novel fluorescent peptide provides a significant pharmacological tool for the exploration and visualization in-situ distribution of α3ß4 nAChRs in different tissues and also assists in clarifying the function.

Imaging and proteomics toolkits for studying organelle contact sites.

Organelle contact sites are regions where two heterologous membranes are juxtaposed by molecular tethering complexes. These contact sites are important in inter-organelle communication and cellular functional integration. However, visualizing these minute foci and identifying contact site proteomes have been challenging. In recent years, fluorescence-based methods have been developed to visualize the dynamic physical interaction of organelles while proximity labeling approaches facilitate the profiling of proteomes at contact sites. In this review, we explain the design principle for these contact site reporters: a dual-organelle interaction mechanism based on how endogenous tethers and/or tethering complexes localize to contact sites. We classify the contact site reporters into three categories: (i) single-protein systems, (ii) two-component systems with activated reporter signal upon organelle proximity, and (iii) reporters for contact site proteomes. We also highlight advanced imaging analysis with high temporal-spatial resolution and the use of machine-learning algorithms for detecting contact sites.

Practical approaches for visualization of endogenous enhancer-promoter interactions in a single nucleus through chromatin labeling.

Recent studies highlight the critical role of nuclear genome organization in regulating gene expression. Dynamic changes in the hierarchical structure of chromatin modulate transcription by influencing the recruitment of transcription factors and altering the epigenetic landscape. Among these regulatory mechanisms, enhancer-promoter (E-P) interactions are of particular importance. Enhancers physically interact with the promoters of target genes, a process mediated by various co-activators, which facilitates the transfer of enhancer-bound transcription factors and ultimately leads to transcriptional bursting. While next-generation sequencing techniques have provided significant insights into the features of E-P interactions, the effects of cell-to-cell heterogeneity and the physical dynamics of these interactions remain poorly understood due to the lack of spatiotemporal information. In this article, we introduce a platform that enables imaging-based approaches to visualize E-P interactions at the single-cell level.

CRISPR/Cas13 sgRNA-Mediated RNA-RNA Interaction Mapping in Live Cells with APOBEC RNA Editing.

Current research on long non-coding RNA (lncRNA) has predominantly focused on identifying their protein partners and genomic binding sites, leaving their RNA partners largely unknown. To address this gap, the study has developed a method called sarID (sgRNA scaffold assisted RNA-RNA interaction detection), which integrates Cas13-based RNA targeting, sgRNA engineering, and proximity RNA editing to investigate lncRNA-RNA interactomes. By applying sarID to the lncRNA NEAT1, over one thousand previously unidentified binding transcripts are discovered. sarID is further expanded to investigate binders of XIST, MALAT1, NBR2, and DANCR, demonstrating its broad applicability in identifying lncRNA-RNA interactions. The findings suggest that lncRNAs may regulate gene expression by interacting with mRNAs, expanding their roles beyond known functions as protein scaffolds, miRNA sponges, or guides for epigenetic modulators. sarID has the potential to be adapted for studying other specific RNAs, providing a novel immunoprecipitation-free method for uncovering RNA partners and facilitating the exploration of the RNA-RNA interactome.

Progress in mass spectrometry approaches to profiling protein-protein interactions in the studies of the innate immune system.

Understanding protein-protein interactions (PPIs) is pivotal for deciphering the intricacies of biological processes. Dysregulation of PPIs underlies a spectrum of diseases, including cancer, neurodegenerative disorders, and autoimmune conditions, highlighting the imperative of investigating these interactions for therapeutic advancements. This review delves into the realm of mass spectrometry-based techniques for elucidating PPIs and their profound implications in biological research. Mass spectrometry in the PPI research field not only facilitates the evaluation of protein-protein interaction modulators but also discovers unclear molecular mechanisms and sheds light on both on- and off-target effects, thus aiding in drug development. Our discussion navigates through six pivotal techniques: affinity purification mass spectrometry (AP-MS), proximity labeling mass spectrometry (PL-MS), cross-linking mass spectrometry (XL-MS), size exclusion chromatography coupled with mass spectrometry (SEC-MS), limited proteolysis-coupled mass spectrometry (LiP-MS), and thermal proteome profiling (TPP).

Oxygen extraction fraction mapping based combining quantitative susceptibility mapping and quantitative blood oxygenation level-dependent imaging model using multi-delay PCASL.

BACKGROUND AND PURPOSE: The oxygen extraction fraction is an essential biomarker for the assessment of brain metabolism. A recently proposed method combined with quantitative susceptibility mapping and quantitative blood oxygen level-dependent magnitude enables noninvasive mapping of the oxygen extraction fraction. Our study investigated the oxygen extraction fraction mapping variations of single-delay and multi-delay arterial spin-labeling. MATERIALS AND METHODS: A total of twenty healthy participants were enrolled. The multi-echo spoiled gradient-echo, multi-delay arterial spin-labeling, and magnetization-prepared rapid gradient echo sequences were acquired at 3.0 T. The mean oxygen extraction fraction was generated under a single delay time of 1780 ms, multi-delay arterial spin-labeling of transit-corrected cerebral blood flow, and multi-delay arterial spin-labeling of arterial cerebral blood volume. The results were compared via paired t tests and the Wilcoxon test. Linear regression analyses were used to investigate the relationships among the oxygen extraction fraction, cerebral blood flow, and venous cerebral blood volume. RESULTS: The oxygen extraction fraction estimate with multi-delay arterial spin-labeling yielded a significantly lower value than that with single-delay arterial spin-labeling. The average values for the whole brain under single-delay arterial spin-labeling, multi-delay arterial spin-labeling of transit-corrected cerebral blood flow, and multi-delay arterial spin-labeling of arterial cerebral blood volume were 41.5 ±â€¯1.7 % (P < 0.05), 41.3 ±â€¯1.9 % (P < 0.001), and 40.9 ±â€¯1.9 % (N = 20), respectively. The oxygen extraction fraction also showed a significant inverse correlation with the venous cerebral blood volume under steady-state conditions when multi-delay arterial spin-labeling was used (r = 0.5834, p = 0.0069). CONCLUSION: These findings suggest that the oxygen extraction fraction is significantly impacted by the arterial spin-labeling methods used in the quantitative susceptibility mapping plus the quantitative blood oxygen level-dependent model, indicating that the differences should be accounted for when employing oxygen extraction fraction mapping based on this model in diseases.

Measurement of absolute abundance of crystallins in human and αA N101D transgenic mouse lenses using 15N-labeled crystallin standards.

Stable isotope labeled standards of all major human lens crystallins were created to measure the abundance of lens endogenous crystallins from birth to adulthood. All major human crystallins (αA, αB, ßA2, ßA3/A1, ßA4, ßB1, ßB2, ßB3, γA, γB, γC, γD, γS) were cloned with N-terminal 6 x His tagged SUMO for ease of purification and the ability to generate natural N-termini by SUMO protease cleavage when producing crystallins for structure/function studies. They were then expressed in 15N-enriched media, quantified by mass spectrometry, and mixed in proportions found in young human lens to act as an artificial lens standard. The absolute quantification method was tested using soluble protein from 5-day, 23-day, 18-month, and 18-year-old human lenses spiked with the 15N artificial lens standard. Proteins were trypsinized, relative ratios of light and heavy labeled peptides determined using high-resolution precursor and data independent MS2 scans, and data analysis performed using Skyline software. Crystallin abundances were measured in both human donor lenses and in transgenic mouse αA N101D cataract lenses. Technical replicates of human crystallin abundance measurements were performed with average coefficients of variation of approximately 2% across all 13 crystallins. αA crystallin comprised 27% of the soluble protein of 5-day-old lens and decreased to 16% by 18-years of age. Over this time period αB increased from 6% to 9% and the αA/αB ratio decreased from 4.5/1 to 2/1. γS-crystallin also increased nearly 2-fold from 7% to 12%, becoming the 3rd most abundant protein in adult lens, while ßB1 increased from 14% to 20%, becoming the most abundant crystallin of adult lens. Minor crystallins ßA2, ßB3, and γA comprised only about 1% each of the newborn lens soluble protein, and their abundance dropped precipitously by adulthood. While 9 of the SUMO tagged crystallins were useful for purification of crystallins for structural studies, γA, γB, γC, and γD were resistant to cleavage by SUMO protease. The abundance of WT and N101D human αA in transgenic mouse lenses was approximately 40-fold lower than endogenous mouse αA, but the deamidation mimic human αA N101D was less soluble than human WT αA. The high content of αA and the transient abundance of ßA2, ßB3, and γA in young lens suggest these crystallins play a role in early lens development and growth. ßB1 becoming the most abundant crystallin may result from its role in promoting higher order ß-crystallin oligomerization in mature lens. The full set of human crystallin expression vectors in the Addgene repository should be a useful resource for future crystallin studies. 15N labeling of these crystallins will be useful to accurately quantify crystallins in lens anatomic regions, as well as measure the composition of insoluble light scattering crystallin aggregates. The standards will also be useful to measure the abundance of crystallins expressed in transgenic animal models.

Proteolysis Assays With Conserved or Aminofluorescein-Labeled Red Blood Cells.

Backgrounds: Various physiological functions and reaction cascades, as well as disease progression in the living systems, are controlled by the activity of specific proteolytic enzymes. We conducted the study to evaluate protease activity by assessing peptide fragments from either conserved or labeled red blood cells (RBCs) with aminofluorescein (AF) in the reaction media. Methods: RBCs were incubated in media containing trypsin. Subsequently, the concentration of peptide fragments in the reaction media, resulted by the digestion with trypsin from conserved cells, was estimated by 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) as an amine-reactive fluorogenic reagent. In a second approach, we conjugated AF to the conserved RBCs and then exposed AF-labeled RBCs to trypsin. This was followed by directly measuring the fluorescence intensity (FI) in the reaction media to estimate the concentration of AF-labeled peptide fragments resulting from the enzyme's activity. Results: Show a concentration- and time-dependent increase in FIs, reflecting the activity of trypsin as a proteolytic enzyme. The FIs increased significantly by 4 to 5 folds in samples treated with different enzyme concentrations, and by over 11 folds after 2 h incubation in media containing a 50 µL trypsin, as evidenced by CBQCA assays. Conclusion: These fast and affordable approaches could be applied with high reliability for the general estimation of protease activity in samples and customized for diagnostic purposes and prognostic evaluation in various diseases.

All-optics technique for monitoring absolute cerebral blood flow: validation against magnetic resonance imaging perfusion.

Significance: The ability to monitor cerebral blood flow (CBF) at the bedside is essential to managing critical-care patients with neurological emergencies. Diffuse correlation spectroscopy (DCS) is ideal because it is non-invasive, portable, and inexpensive. We investigated a near-infrared spectroscopy (NIRS) approach for converting DCS measurements into physiological units of blood flow. Aim: Using magnetic resonance imaging perfusion as a reference, we investigated the accuracy of absolute CBF measurements from a bolus-tracking NIRS method that used transient hypoxia as a flow tracer and hypercapnia-induced increases in CBF measured by DCS. Approach: Twelve participants (7 female, 28 ± 6 years) completed a hypercapnia protocol with simultaneous CBF recordings from DCS and arterial spin labeling (ASL). Nine participants completed the transient hypoxia protocol while instrumented with time-resolved NIRS. The estimate of baseline CBF was subsequently used to calibrate hypercapnic DCS data. Results: Moderately strong correlations at baseline ( slope = 0.79 and R 2 = 0.59 ) and during hypercapnia ( slope = 0.90 and R 2 = 0.58 ) were found between CBF values from calibrated DCS and ASL (range 34 to 85 mL / 100 g / min ). Conclusions: Results demonstrated the feasibility of an all-optics approach that can both quantify CBF and perform continuous perfusion monitoring.

Choice architecture promotes sustainable choices in online food-delivery apps.

Greenhouse gas emissions from the food system constitute about one-third of the global total, hence mitigation in this sphere of human activity is a vital goal for research and policy. This study empirically tests the effectiveness of different interventions to reduce the carbon footprint of food choices made on food-delivery apps, using an incentive-compatible online randomized controlled trial with 4,008 participants. The experiment utilized an interactive web platform that mimics popular online food-delivery platforms (such as Just Eat) and included three treatment conditions: a sign-posted meat tax, a carbon-footprint label, and a choice-architecture intervention that changed the order of the menu so that the lowest carbon-impact restaurants and dishes were presented first. Results show that only the choice-architecture nudge significantly reduced the average meal carbon footprint-by 0.3 kg/CO2e per order (12%), driven by a 5.6 percentage point (13%) reduction in high-carbon meal choices. Moreover, we find evidence of significant health and well-being co-benefits. Menu repositioning resulted in the average meal order having greater nutritional value and fewer calories, whilst significantly increasing self-reported satisfaction with the meal choice. Simple back-of-the-envelope calculations suggest that menu repositioning would be a highly cost-effective policy instrument if implemented at scale, with the return on investment expected to be in the range of £1.28 to £3.85 per metric ton of avoided CO2 emissions, depending on implementation costs.

Covalent modification of protein by chemical probe in living cells for structural and interaction studies.

Cellular activities are predominantly carried out by proteins that can dynamically adopt different structural conformations and differentially interact with other biomolecules according to cellular needs. Chemical probes are small molecules used to selectively interact and modulate the activities of specific proteins to study their functions such as the validation of potential drug targets. The remarkable performance of AlphaFold algorithms in the prediction of protein structures has pivoted interest toward elucidating the intracellular dynamics of protein structural conformation where covalent modification of proteins by chemical probes could be used to shed light upon. However, due to the barrier to entry by cell membrane and the general unfavorable reactive conditions of the intracellular environment, most studies using reactive chemical probes are still conducted on purified proteins and cell lysates. Nevertheless, recent progresses have been made in designing chemical probes with improved membrane permeability, stability and reactivity. This paper surveys the literature on recent advancements in membrane-permeable chemical probes and their applications with protein mass spectrometry for the intracellular studies of protein structural conformations and biomolecular interactions.

Soil carbon and nitrogen cycling at the atmosphere-soil interface: Quantifying the responses of biocrust-soil interactions to global change.

In drylands, where water scarcity limits vascular plant growth, much of the primary production occurs at the soil surface. This is where complex macro- and microbial communities, in an intricate bond with soil particles, form biological soil crusts (biocrusts). Despite their critical role in regulating C and N cycling in dryland ecosystems, there is limited understanding of the fate of biologically fixed C and N from biocrusts into the mineral soil, or how climate change will affect C and N fluxes between the atmosphere, biocrusts, and subsurface soils. To address these gaps, we subjected biocrust-soil systems to experimental warming and drought under controlled laboratory conditions, monitored CO2 fluxes, and applied dual isotopic labeling pulses (13CO2 and 15N2). This allowed detailed quantification of elemental pathways into specific organic matter (OM) pools and microbial biomass via density fractionation and phospholipid fatty acid analyses. While biocrusts modulated CO2 fluxes regardless of the temperature regime, drought severely limited their photosynthetic C uptake to the extent that the systems no longer sustained net C uptake. Furthermore, the effect of biocrusts extended into the underlying 1 cm of mineral soil, where C and N accumulated as mineral-associated OM (MAOM<63µm). This was strongly associated with increased relative dominance of fungi, suggesting that fungal hyphae facilitate the downward C and N translocation and subsequent MAOM formation. Most strikingly, however, these pathways were disrupted in systems exposed to warming, where no effects of biocrusts on the elemental composition of the underlying soil nor on MAOM were determined. This was further associated with reduced net biological N fixation under combined warming and drought, highlighting how changing climatic conditions diminish some of the most fundamental ecosystem functions of biocrusts, with detrimental repercussions for C and N cycling and the persistence of soil organic matter pools in dryland ecosystems.

En regiones áridas, donde la sequía limita el crecimiento de plantas vasculares, gran parte de la producción primaria ocurre en la superficie del suelo. En este lugar, complejas comunidades microbianas, estrechamente ligadas a partículas del suelo, forman costras biológicas (conocidas también como biocostras). Aunque estas biocostras son cruciales para regular los ciclos del carbono (C) y nitrógeno (N) en ecosistemas áridos, aún existe una comprensión limitada del destino hacia el suelo mineral del C y N fijados biológicamente desde las biocostras, o sobre cómo el cambio climático afectará los flujos de C y N entre la atmósfera, las biocostras y los suelos subsuperficiales. Para abordar estas brechas, sometimos sistemas de biocostra y suelo a aumentos de temperatura y sequía experimentales en condiciones controladas de laboratorio, donde monitoreamos los flujos de CO2 y aplicamos pulsos de etiquetado isotópico dual (13CO2 y 15N2). Esto permitió una cuantificación detallada de las vías de incorporación de los elementos en grupos específicos de materia orgánica (MO) y biomasa microbiana mediante fraccionamiento por densidad y análisis de ácidos grasos de fosfolípidos (PLFA). Si bien las biocostras modularon los flujos de CO2 independientemente del régimen de la temperatura, la sequía restringió severamente la captación fotosintética de C hasta el punto de que los sistemas ya no mantuvieron la absorción neta de C. Además, el efecto de las biocostras se extendió hasta 1 cm del suelo bajo esta, donde el C y el N se acumularon como MO asociada a minerales (MAOM<63µm). Esto se relaciona estrechamente con un aumento en la dominancia relativa de hongos, lo que sugiere que las hifas de los hongos facilitan la translocación descendente de C y N y subsecuentemente la formación de MAOM. Sin embargo, lo más sorprendente es que estas vías se vieron interrumpidas en sistemas expuestos al aumento de temperatura, donde no se determinaron efectos de las biocostras en la composición elemental del suelo subyacente ni en la MAOM. Esto se asoció con una reducción de la fijación biológica neta de N bajo el efecto combinado del aumento de la temperatura y la sequía, destacando cómo las condiciones climáticas cambiantes disminuyen algunas de las funciones ecosistémicas más fundamentales de las biocostras, con repercusiones perjudiciales para el ciclo de C y N y la persistencia de los depósitos de MOS en los ecosistemas áridos.

Detection of Tn-antigen in breast and prostate cancer models by VVL-labeled red dye-doped nanoparticles.

Aim: Fluorescence detection of breast and prostate cancer cells expressing Tn-antigen, a tumor marker, with Vicia villosa lectin (VVL)-labeled nanoparticles.Materials & methods: Breast and prostate cancer cells engineered to express high levels of Tn-antigen and non-engineered controls were incubated with VVL-labeled or unlabeled red dye-doped silica-coated polystyrene nanoparticles. The binding to cells was studied with flow cytometry, confocal microscopy, and electron microscopy.Results: Flow cytometry showed that the binding of VVL-labeled nanoparticles was significantly higher to Tn-antigen-expressing cancer cells than controls. Confocal microscopy demonstrated that particles bound to the cell surface. According to the correlative light and electron microscopy the particles bound mostly as aggregates.Conclusion: VVL-labeled nanoparticles could provide a new tool for the detection of Tn-antigen-expressing breast and prostate cancer cells.

[Box: see text].