Probing the Effect of Acidosis on Tether-Mode Mechanotransduction of Proprioceptors.

Proprioceptors are low-threshold mechanoreceptors involved in perceiving body position and strain bearing. However, the physiological response of proprioceptors to fatigue- and muscle-acidosis-related disturbances remains unknown. Here, we employed whole-cell patch-clamp recordings to probe the effect of mild acidosis on the mechanosensitivity of the proprioceptive neurons of dorsal root ganglia (DRG) in mice. We cultured neurite-bearing parvalbumin-positive (Pv+) DRG neurons on a laminin-coated elastic substrate and examined mechanically activated currents induced through substrate deformation-driven neurite stretch (SDNS). The SDNS-induced inward currents (ISDNS) were indentation depth-dependent and significantly inhibited by mild acidification (pH 7.2~6.8). The acid-inhibiting effect occurred in neurons with an ISDNS sensitive to APETx2 (an ASIC3-selective antagonist) inhibition, but not in those with an ISNDS resistant to APETx2. Detailed subgroup analyses revealed ISDNS was expressed in 59% (25/42) of Parvalbumin-positive (Pv+) DRG neurons, 90% of which were inhibited by APETx2. In contrast, an acid (pH 6.8)-induced current (IAcid) was expressed in 76% (32/42) of Pv+ DRG neurons, 59% (21/32) of which were inhibited by APETx2. Together, ASIC3-containing channels are highly heterogenous and differentially contribute to the ISNDS and IAcid among Pv+ proprioceptors. In conclusion, our findings highlight the importance of ASIC3-containing ion channels in the physiological response of proprioceptors to acidic environments.

Comparative of clinical performance between next-generation sequencing and standard blood culture diagnostic method in patients suffering from sepsis.

BACKGROUND: Next-generation sequencing (NGS) is a massively unbiased sequencing technology. The objective of this study was to evaluate the performance of NGS-based approach in the detection of microorganisms from septic patients and compare with results of blood culture (BC). METHODS: The observational and non-interventional study was conducted from April 2019 to August 2019. RESULTS: A total of 96 sets of BC and 48 NGS results obtained from 48 septic patients were analyzed in this study. Thirty-two microorganisms (27 bacteria, 3 fungi and 2 viral) were detected by NGS in 23 (47.9%) patients; and 18 bacteria in 18 (37.5%) patients by BC. Exclusion of skin commensals, the positivity of NGS and BC was 62.5% and 14.5%, respectively (P < 0.001). Microorganisms identified by NGS demonstrated positive agreement with BC in 12 (25%) patients, including concordant results in 11 (22.9%) cases, and discrepancy results in 1 (2%). Of 11 patients with concordant results, 4 had additional microorganisms detected by NGS. NGS-positive but BC-negative was found in 9 (18.7%) patients. Using NGS, difficult-to-culture micro-organisms such as Pneumocystic jirovecii was identified in 2 patients, and Leptospira interrogans in one. Six (12.5%) patients with BC-positive but NGS-negative, whereas skin commensals were isolated in 4 (66.6%) cases. The number of patients that were positive by BC only increase from 29% to 47.9% when combining NGS and BC analyses (P = 0.033). CONCLUSIONS: Our study support the advantage of NGS for the diagnosis of infecting microorganisms in sepsis, especially for microorganisms that are currently difficult or impossible to culture.

Clinical Application of Metagenomic Next-Generation Sequencing in Patients with Hematologic Malignancies Suffering from Sepsis.

BACKGROUND: Sepsis remains a common but fatal complication among patients with immune suppression. We aimed to investigate the performance of metagenomic next-generation sequencing (mNGS) compared with standard microbiological diagnostics in patients with hematologic malignancies. METHODS: We performed a prospective study from June 2019 to December 2019. Adult patients with hematologic malignancies and a clinical diagnosis of sepsis were enrolled. Conventional diagnostic methods included blood cultures, serum galactomannan for Aspergillus, cryptococcal antigen and cytomegalovirus (CMV) viral loads. Blood samples for mNGS were collected within 24 h after hypotension developed. RESULTS: Of 24 patients enrolled, mNGS and conventional diagnostic methods (blood cultures, serology testing and virus RT-PCR) reached comparable positive results in 9 cases. Of ten patients, mNGS was able to identify additional pathogens compared with conventional methods; most of the pathogens were virus. CONCLUSION: Our results show that mNGS may serve as adjunctive diagnostic tool for the identification of pathogens of hematologic patients with clinically sepsis.

The Extracellular Zn2+ Concentration Surrounding Excited Neurons Is High Enough to Bind Amyloid-ß Revealed by a Nanowire Transistor.

The Zn2+ stored in the secretory vesicles of glutamatergic neurons is coreleased with glutamate upon stimulation, resulting in the elevation of extracellular Zn2+ concentration (CZn2+ex). This elevation of CZn2+ex regulates the neurotransmission and facilitates the fibrilization of amyloid-ß (Aß). However, the exact CZn2+ex surrounding neurons under (patho)physiological conditions is not clear and the connection between CZn2+ex and the Aß fibrilization remains obscure. Here, a silicon nanowire field-effect transistor (SiNW-FET) with the Zn2+ -sensitive fluorophore, FluoZin-3 (FZ-3), to quantify the CZn2+ex in real time is modified. This FZ-3/SiNW-FET device has a dissociation constant of ≈12 × 10-9 m against Zn2+ . By placing a coverslip seeded with cultured embryonic cortical neurons atop an FZ-3/SiNW-FET, the CZn2+ex elevated to ≈110 × 10-9 m upon stimulation with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Blockers against the AMPA receptor or exocytosis greatly suppress this elevation, indicating that the Zn2+ stored in the synaptic vesicles is the major source responsible for this elevation of CZn2+ex. In addition, a SiNW-FET modified with Aß could bind Zn2+ with a dissociation constant of ≈633 × 10-9 m and respond to the Zn2+ released from AMPA-stimulated neurons. Therefore, the CZn2+ex can reach a level high enough to bind Aß and the Zn2+ homeostasis can be a therapeutic strategy to prevent neurodegeneration.

Calmodulin modulates the Ca2+-dependent inactivation and expression level of bovine CaV2.2 expressed in HEK293T cells.

Ca2+ influx through voltage-gated Ca2+ channels (CaVs) at the plasma membrane is the major pathway responsible for the elevation of the intracellular Ca2+ concentration ([Ca2+]i), which activates various physiological activities. Calmodulin (CaM) is known to be involved in the Ca2+-dependent inactivation (CDI) of several types of CaVs; however, little is known about how CaM modulates CaV2.2. Here, we expressed CaV2.2 with CaM or CaM mutants with a Ca2+-binding deficiency in HEK293T cells and measured the currents to characterize the CDI. The results showed that CaV2.2 displayed a fast inactivation with Ca2+ but not Ba2+ as the charge carrier; when CaV2.2 was co-expressed with CaM mutants with a Ca2+-binding deficiency, the level of inactivation decreased. Using glutathione S-transferase-tagged CaM or CaM mutants as the bait, we found that CaM could interact with the intracellular C-terminal fragment of CaV2.2 in the presence or absence of Ca2+. However, CaM and its mutants could not interact with this fragment when mutations were generated in the conserved amino acid residues of the CaM-binding site. CaV2.2 with mutations in the CaM-binding site showed a greatly reduced current that could be rescued by CaM12 (Ca2+-binding deficiency at the N-lobe) overexpression; in addition, CaM12 enhanced the total expression level of CaV2.2, but the ratio of CaV2.2 present in the membrane to the total fraction remained unchanged. Together, our data suggest that CaM, with different Ca2+-binding abilities, modulates not only the inactivation of CaV2.2 but also its expression to regulate Ca2+-related physiological activities.