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BackgroundNeurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ is critical to facilitate discovery and validation of pathogenic molecular pathways. Immunostaining methods enable in situ detection of specific targets. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue adhesion and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Furthermore, stripping techniques require antibody-specific optimization, validation, and quality control steps.New methodAiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives based on extensive testing.ResultsA protocol using β-mercaptoethanol and SDS consistently enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue adhesion and antigen integrity in human postmortem tissue.Comparison with existing methodsOur proposed method is optimal for standard lab settings and shows consistent efficacy despite the intricacies of suboptimal human postmortem tissue and the need to strip markers bound to highly aggregated proteins. Additionally, it incorporates quality control steps to validate antibody stripping.ConclusionsMultiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters during optimization and validation phases of experiments is prudent.

Although biochar application to soils has been found to increase soil quality and crop yield, the biochar dispersion extent and its impacts on native soil organic carbon (SOC) has received relatively little attention. Here, the vertical and lateral migration of fine, intermediate and coarse-sized biochar (<0.5, 0.5–1 and 1–5 mm, respectively), applied at low and high doses (1.5–2 and 3–4% w/w, respectively), was tracked using stable isotope methods, along with its impact on native SOC stocks. Biochar was homogeneously mixed into the surface layer (0–7 cm depth) of a loamy sandy Acrisol in Zambia. After 4.5 y, 38–75% of the biochar carbon (BC) was lost from the applied layer and 4–25% was detected in lower soil layers (7–30 cm). Estimating BC mineralization to be no more than 8%, 25–60% was likely transported laterally out of the experimental plots. This conclusion was supported by observations of BC in the control plot and in soils up to 2 m outside of the experimental plots. These processes were likely progressive as recovery of BC in similar plots 1 year after application was greater in both surface and lower soil layers than after 4.5 y. Fine and intermediate-sized BC displayed the greatest downward migration (25.3 and 17.9%, respectively), particularly when applied at lower doses, suggesting its movement through soil inter-particle spaces. At higher dosages, fine and intermediate-sized particles may have clogged pore, so coarse biochar displayed the greatest downward migration when biochar was applied at higher doses. In the BC treatment plot soil profiles, native SOC stocks were reduced by 2.8 to 24.5% (18.4% on average), i.e. positive priming. However, some evidence suggested that the soils may switch to negative priming over time. The dispersion of biochar in soil should be considered when evaluating biochar's agronomic benefits and environmental effects. [ABSTRACT FROM AUTHOR]

The recently developed time-of-flight aerosol chemical speciation monitor with a capture vaporizer and a PM 2.5 aerodynamic lens (TOF-ACSM-CV-PM 2.5) aims to improve the collection efficiency and chemical characterization of aerosol particles with a diameter smaller than 2.5 µm. In this study, comprehensive cross-comparisons were performed between real-time online measurements and offline filter analysis with 24 h collection time. The goal was to evaluate the capabilities of the TOF-ACSM-CV-PM 2.5 lens, as well as the accuracy of the TOF-ACSM-CV-PM 2.5. The experiments were conducted at Cabauw Experimental Site for Atmospheric Research (CESAR) during the RITA-2021 campaign. The non-refractory fine particulate matter (PM 1.0 and PM 2.5) was measured by two collocated TOF-ACSM-CV-PM 2.5 instruments by placing them behind a PM 2.5 and PM 1.0 inlet, respectively. A comparison between the ACSMs and PM 2.5 and PM 1.0 filter samples showed a much better accuracy than ±30 % less given in the previous reports, with average differences less than ±10 % for all inorganic chemical species. In addition, the ACSMs were compared to the Monitoring Instrument for Aerosol and Gas (MARGA) (slope between 0.78 and 0.97 for inorganic compounds, R2≥ 0.93) and a mobility particle size spectrometer (MPSS), measuring the particle size distribution from around 10 to 800 nm (slope was around 1.00, R2= 0.91). The intercomparison of the online measurements and the comparison between the online and offline measurements indicated a low bias (< 10 % for inorganic compounds) and demonstrated the high accuracy and stability of the TOF-ACSM-CV-PM 2.5 lens for the atmospheric observations of particle matter. The two ACSMs exhibited an excellent agreement, with differences less than 7 %, which allowed a quantitative estimate of PM 1.0 vs. PM 2.5 chemical composition. The result showed that the PM 1.0 accounted for about 70 %–80 % of the PM 2.5 on average. The NO 3 mass fraction increased, but the organic carbon (OC) mass fraction decreased from PM 1.0 to PM 2.5 , indicating the size dependence on chemical composition. [ABSTRACT FROM AUTHOR]

The mu and delta types of opioid receptors form heteromers that exhibit pharmacological and functional properties distinct from those of homomeric receptors. To characterize these complexes in the brain, we generated antibodies that selectively recognize the mu-delta heteromer and blocked its in vitro signaling. With these antibodies, we showed that chronic, but not acute, morphine treatment caused an increase in the abundance of mu-delta heteromers in key areas of the central nervous system that are implicated in pain processing. Because of its distinct signaling properties, the mu-delta heteromer could be a therapeutic target in the treatment of chronic pain and addiction.

Pain signals are transmitted by multisynaptic glutamatergic pathways. Their first synapse between primary nociceptors and excitatory spinal interneurons gates the sensory load. In this pathway, glutamate release is orchestrated by [Ca.sup.2+]-sensor proteins, with N-terminal EF-hand [Ca.sup.2+]-binding protein 2 (NECAB2) being particular abundant. However, neither the importance of [NECAB2.sup.+] neuronal contingents in dorsal root ganglia (DRGs) and spinal cord nor the function determination by NECAB2 has been defined. A combination of histochemical analyses and single- cell RNA-sequencing showed NECAB2 in small- and medium-sized C- and A[delta] D-hair low-threshold mechanoreceptors in DRGs, as well as in protein kinase C [gamma] excitatory spinal interneurons. NECAB2 was downregulated by peripheral nerve injury, leading to the hypothesis that NECAB2 loss of function could limit pain sensation. Indeed, [Necab2.sup.-/-] mice reached a pain-free state significantly faster after peripheral inflammation than did WT littermates. Genetic access to transiently activated neurons revealed that a mediodorsal cohort of [NECAB2.sup.+] neurons mediates inflammatory pain in the mouse spinal dorsal horn. Here, besides dampening excitatory transmission in spinal interneurons, NECAB2 limited pronociceptive brain-derived neurotrophic factor (BDNF) release from sensory afferents. Hoxb8-dependent reinstatement of NECAB2 expression in [Necab2.sup.-/-] mice then demonstrated that spinal and DRG NECAB2 alone could control inflammation-induced sensory hypersensitivity. Overall, we identify NECAB2 as a critical component of pronociceptive pain signaling, whose inactivation offers substantial pain relief., Introduction Acute pain (nociception) is a physiologically relevant sensation aimed at alerting an individual to, and thus limiting, tissue damage upon contact with harmful stimuli. In turn, chronic pain is [...]

The recently developed time of flight-aerosol chemical speciation monitor with the capture vaporizer and a PM2.5 aerodynamic lens (TOF-ACSM-CV-PM2.5) aims to improve the collection efficiency and chemical characterization of aerosol particles with diameter smaller than 2.5µm. In this study, comprehensive cross-comparisons were performed between real-time online measurements and offline filter analysis with 24-hour collection time. The goal was to evaluate the capabilities of the TOFACSM-CV-PM2.5 lens, as well as the accuracy of the TOF-ACSM-CV-PM2.5. The experiments were conducted at Cabauw Experimental Site for Atmospheric Research (CESAR) during the RITA-2021 campaign. The non-refractory fine particulate matter PM1.0 and PM2.5 were measured by two co-located TOF-ACSM-CV-PM2.5 by placing them behind a PM2.5 and PM1.0 inlet, respectively. A comparison between the ACSMs and PM2.5 and PM1.0 filter samples showed a much better accuracy than ±30% less given in the previous reports, with average differences less than ± 10% for all inorganic chemical species. In addition, the ACSMs were compared to a Monitoring Instrument for Aerosol and Gas (MARGA) (slope between 0.78 - 0.97 for inorganic compounds, R2 = 0.93), and a Mobility Particle Size Spectrometer (MPSS) measuring the particle size distribution from around 10 to 800 nm (slope was around 1.00, R2 = 0.91). The intercomparison of the online measurements and the comparison between the online and offline measurements indicated a low bias (< 10% for inorganic compounds) and demonstrated the high accuracy and stability of the TOF-ACSM-CV-PM2.5 lens for the atmospheric observations of particle matters. The two ACSMs exhibited an excellent agreement, with differences less than 7%, which allowed a quantitative estimate of PM1.0 vs PM2.5 chemical composition. The result showed that the PM1.0 accounted for about 70-80% of the PM2.5 on average. The NO3 mass fraction increased but the OC mass fraction decreased from PM1.0 to PM2.5, indicating the size30 dependence on chemical composition. [ABSTRACT FROM AUTHOR]