The Effect of Lung Resection for NSCLC on Circulating Immune Cells: A Pilot Study.

This pilot study sought to evaluate the circulating levels of immune cells, particularly regulatory T-cell (Treg) subsets, before and after lung resection for non-small cell lung cancer. Twenty-five patients consented and had specimens collected. Initially, peripheral blood of 21 patients was collected for circulating immune cell studies. Two of these patients were excluded due to technical issues, leaving 19 patients for the analyses of circulating immune cells. Standard gating and high-dimensional unsupervised clustering flow cytometry analyses were performed. The blood, tumors and lymph nodes were analyzed via single-cell RNA and TCR sequencing for Treg analyses in a total of five patients (including four additional patients from the initial 21 patients). Standard gating flow cytometry revealed a transient increase in neutrophils immediately following surgery, with a variable neutrophil-lymphocyte ratio and a stable CD4-CD8 ratio. Unexpectedly, the total Treg and Treg subsets did not change with surgery with standard gating in short- or long-term follow-up. Similarly, unsupervised clustering of Tregs revealed a dominant cluster that was stable perioperatively and long-term. Two small FoxP3hi clusters slightly increased following surgery. In the longer-term follow-up, these small FoxP3hi Treg clusters were not identified, indicating that they were likely a response to surgery. Single-cell sequencing demonstrated six CD4+FoxP3+ clusters among the blood, tumors and lymph nodes. These clusters had a variable expression of FoxP3, and several were mainly, or only, present in tumor and lymph node tissue. As such, serial monitoring of circulating Tregs may be informative, but not completely reflective of the Tregs present in the tumor microenvironment.

Transcript decay mediated by RNase III in Borrelia burgdorferi.

The causative agent of Lyme disease, Borrelia burgdorferi, requires shifts in gene expression to undergo its natural enzootic cycle between tick and vertebrate hosts. mRNA decay mechanisms play significant roles in governing gene expression in other bacteria, but are not yet characterized in B. burgdorferi. RNase III is an important enzyme in processing ribosomal RNA, but it also plays a role in mRNA decay in many bacteria. We compared RNA decay profiles and steady-state abundances of transcripts in wild-type Borrelia burgdorferi strain B31 and in an RNase III null (rnc-) mutant. Transcripts encoding RNA polymerase subunits (rpoA and rpoS), ribosomal proteins (rpsD, rpsK, rpsM, rplQ, and rpsO), a nuclease (pnp), a flagellar protein (flaB), and a translational regulator (bpuR) decayed more rapidly in the wild-type strain than in the slow growing rnc- mutant indicating that RNA turnover is mediated by RNase III in the bacterium that causes Lyme disease. Additionally, in wild type bacteria, RNA decay rates of rpoS, rpoN, ospA, ospC, bpuR and dbpA transcripts are only modestly affected by changes in the osmolarity.

Related neuropeptides use different balances of unitary mechanisms to modulate the cardiac neuromuscular system in the American lobster, Homarus americanus.

To produce flexible outputs, neural networks controlling rhythmic motor behaviors can be modulated at multiple levels, including the pattern generator itself, sensory feedback, and the response of the muscle to a given pattern of motor output. We examined the role of two related neuropeptides, GYSDRNYLRFamide (GYS) and SGRNFLRFamide (SGRN), in modulating the neurogenic lobster heartbeat, which is controlled by the cardiac ganglion (CG). When perfused though an isolated whole heart at low concentrations, both peptides elicited increases in contraction amplitude and frequency. At higher concentrations, both peptides continued to elicit increases in contraction amplitude, but GYS caused a decrease in contraction frequency, while SGRN did not alter frequency. To determine the sites at which these peptides induce their effects, we examined the effects of the peptides on the periphery and on the isolated CG. When we removed the CG and stimulated the motor nerve with constant bursts of stimuli, both GYS and SGRN increased contraction amplitude, indicating that each peptide modulates the muscle or the neuromuscular junction. When applied to the isolated CG, neither peptide altered burst frequency at low peptide concentrations; at higher concentrations, SGRN decreased burst frequency, whereas GYS continued to have no effect on frequency. Together, these data suggest that the two peptides elicit some of their effects using different mechanisms; in particular, given the known feedback pathways within this system, the importance of the negative (nitric oxide) relative to the positive (stretch) feedback pathways may differ in the presence of the two peptides.

The neuromuscular transform of the lobster cardiac system explains the opposing effects of a neuromodulator on muscle output.

Motor neuron activity is transformed into muscle movement through a cascade of complex molecular and biomechanical events. This nonlinear mapping of neural inputs to motor behaviors is called the neuromuscular transform (NMT). We examined the NMT in the cardiac system of the lobster Homarus americanus by stimulating a cardiac motor nerve with rhythmic bursts of action potentials and measuring muscle movements in response to different stimulation patterns. The NMT was similar across preparations, which suggested that it could be used to predict muscle movement from spontaneous neural activity in the intact heart. We assessed this possibility across semi-intact heart preparations in two separate analyses. First, we performed a linear regression analysis across 122 preparations in physiological saline to predict muscle movements from neural activity. Under these conditions, the NMT was predictive of contraction duty cycle but was unable to predict contraction amplitude, likely as a result of uncontrolled interanimal variability. Second, we assessed the ability of the NMT to predict changes in motor output induced by the neuropeptide C-type allatostatin. Wiwatpanit et al. (2012) showed that bath application of C-type allatostatin produced either increases or decreases in the amplitude of the lobster heart contractions. We show that an important component of these preparation-dependent effects can arise from quantifiable differences in the basal state of each preparation and the nonlinear form of the NMT. These results illustrate how properly characterizing the relationships between neural activity and measurable physiological outputs can provide insight into seemingly idiosyncratic effects of neuromodulators across individuals.