Insights into the dynamic control of breathing revealed through cell-type-specific responses to substance P

The rhythm generating network for breathing must continuously adjust to changing metabolic and behavioral demands. Here, we examined network-based mechanisms in the mouse preBötzinger complex using substance P, a potent excitatory modulator of breathing frequency and stability, as a tool to dissect network properties that underlie dynamic breathing. We find that substance P does not alter the balance of excitation and inhibition during breaths or the duration of the resulting refractory period. Instead, mechanisms of recurrent excitation between breaths are enhanced such that the rate that excitation percolates through the network is increased. We propose a conceptual framework in which three distinct phases of inspiration, the burst phase, refractory phase, and percolation phase, can be differentially modulated to control breathing dynamics and stability. Unraveling mechanisms that support this dynamic control may improve our understanding of nervous system disorders that destabilize breathing, many of which involve changes in brainstem neuromodulatory systems.

Insights into the dynamic control of breathing revealed through cell-type-specific responses to substance P

The rhythm generating network for breathing must continuously adjust to changing metabolic and behavioral demands. Here, we examine network-based mechanisms in the mouse preBӧtzinger complex using substance P, a potent excitatory modulator of breathing frequency and stability, as a tool to dissect network properties that underlie dynamic breathing. We find that substance P does not alter the balance of excitation and inhibition during breaths or the duration of the resulting refractory period. Instead, mechanisms of recurrent excitation between breaths are enhanced such that the rate that excitation percolates through the network is increased. Based on our results, we propose a conceptual framework in which three distinct phases, the inspiratory phase, refractory phase, and percolation phase, can be differentially modulated to influence breathing dynamics and stability. Unravelling mechanisms that support this dynamic control may improve our understanding of nervous system disorders that destabilize breathing, many of which are associated with changes in brainstem neuromodulatory systems.

Effect of external lighting on biopotential of maize leaves caused by pulsed temperature stimulation

Study of electrophysiological indicators of the condition and behavior of plants has become more important in the development of farming activities and the search for effective ways to improve the productivity of crops. The influence of external light on the adaptive ability of corn leaf cells to rhythmic cold stimulation was determined experimentally. The method of rhythmic cold stimulation is not adequate for the studied plants, but its application allows us to evaluate the stability of plant cells to external stimuli. The method consists in repeating irritation during the time period of less duration than the relative refractory phase, which causes a response of less than the previous amplitude. Because of this in the system there is a negative feedback that leads to stabilization of the amplitude of biopotentials that are registered. Rhythmic cold stimulation was applied to the leaf with the help of a quick-response thermostimulator. Rhythmic cold stimuli and settings of pulses were set by computer software. Cooling temperature was controlled using miniature differential thermocouple. Potentials of the leaf surface were diverted by an unpolarized macroelectrode and after a preamplifier fed to the input of the USB oscilloscope connected to the computer. Analysis of the results of experiments was performed using automated developed software. As a result, we experimentally established that rhythmic stimulation of leaves by cold leads to stabilization of responding potential. The level of stabilization depends on the frequency of cold stimuli and describes the adaptive properties of the system causing the biopotential. We found that the absence of photosynthesis when there is a deficit outdoor lighting leads to a significant increase in the average level of stabilized responses, indicating increased stability of the system to external influences. The maximum of this increase fell on the fourth day. This increase is likely to be due to the restructuring of functional ion transport through cell membranes, generating potentials registered. In the interval 4–9th days there was a significant decrease in stabilization, probably due to adaptation of plant cells to a lack of light, or depletion of ATP, which provides the active transport of ions across the cell membrane.

Identification of Somatic Mutation Contributing to Chemotherapy Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease with various underlying genetic alterations. After conventional induction chemotherapy with cytarabine and anthracycline, approximately 20% of AML patients fail to achieve complete remission, associated with poor prognosis. Despite the recent identification of novel recurrent driver mutations and advances in the understanding of the molecular pathogenesis, little is known about the relationship between genetic abnormalities and chemoresistance in AML. Herein, we aimed to reveal somatic mutations contributing to chemoresistance in primary refractory AML patients. Firstly, we performed whole-exome sequencing (WES) analysis using paired samples at diagnosis and refractory phase from 6 primary refractory AML patients. All patients were failed to achieve complete remission by at least two cycles of standard induction chemotherapy with cytarabine and idarubicin. Buccal mucosa or CD3+ T cells were used as germline control. All single nucleotide variants detected by WES were validated by targeted sequencing. As a result, we identified total of 50 somatic mutations including previously described mutated genes in AML patients such as ASXL1, ASXL2, BCOR, DSCAM, GBP4, KCNH2, KIT, LRP1B, MUC5B, PTPRN, PTPN11, SMAD9, U2AF1, and VASH1. The average number of somatic mutations was 8.3 per case (range 2-17), and there were no recurrent mutations among these 6 patients. Next, targeted sequencing of all coding sequence (CDS) of these 50 genes, in which mutations were found by WES, was performed using 18 primary refractory AML samples at refractory phase. Interestingly, we identified BCOR nonsense or frameshift mutations in 4 samples (22%). Although BCOR disruptive mutations were known to be associated with various hematologic malignancies including AML, the frequency of BCOR mutations in our cohort was higher than that of previously reported historical controls from TCGA of 200 de novo AML samples. Therefore, we performed targeted sequencing of all CDS of BCOR using additional 50 samples at diagnosis including 15 primary refractory AML and 35 responder AML who achieved CR after 1 or 2 cycles of induction chemotherapy. As a result, BCOR disruptive mutations were detected in 3/15 (20%) cases in primary refractory AML samples and 1/35 (3%) cases in responder AML samples, revealing that BCOR mutations were enriched in refractory phase samples. These results indicate that BCOR disruptive mutations are related to chemoresistance in AML. To explore the biological effect of BCOR disruptive mutations in chemoresistance, we generated BCOR knockout THP-1 human AML cell lines using CRISPR/Cas9 system. When we examined the effect of BCOR nullizygosity on cell proliferation, BCOR-null THP-1 cells showed decreased proliferation capacity compared with BCOR-intact control cells in liquid culture condition. Importantly, in vitro administration with cytarabine or idarubicin showed decreased percentage of apoptotic cells in BCOR-knockout THP-1 cells compared with BCOR-intact cells. Finally, we established murine subcutaneous xenograft model of BCOR-null THP-1 cells or BCOR-intact control cells to investigate the chemoresistance nature of BCOR nullzygosity in vivo. As expected, BCOR-null mice showed increased relative tumor volume compared with BCOR-intact control mice after idarubicin administration, consistent with low complete remission rate of BCOR-mutated AML in human cases. Taken together, our findings demonstrated that BCOR disruptive mutations are frequently associated with primary refractory AML and confer resistance to conventional chemotherapy with cytarabine and anthracycline, highlighting the clinical and biological significance of BCOR mutation in AML. Further understanding of the molecular basis of chemoresistance in BCOR-mutated AML may provide novel insights into mechanism of chemoresistance and new therapeutic target of primary refractory AML. Disclosures Kataoka: Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria.