Stimulation Conditions Research Articles

Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies.

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC+ format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rβγ stimulation, whereas the L45E mutation optimized IL-15Rβγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC+ constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC+ constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC+ constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

Transcranial focused ultrasound stimulation in the infralimbic cortex facilitates extinction of conditioned fear in rats

Transcranial focused ultrasound (tFUS) neuromodulation emerges as a promising non-invasive approach for improving neurological conditions. Extinction of conditioned fear has served as a prime model for exposure-based therapies for anxiety disorders. We investigated whether tFUS stimulation to a critical brain area, the infralimbic subdivision of the prefrontal cortex (IL), could facilitate fear extinction using rats. In a series of experiments, tFUS was delivered to the IL of a freely-moving rat and compared to sham stimulation (tFUS vs. SHAM). Initially, Fos expression in the IL was measured shortly after the stimulation. The results show that Fos expression was significantly increased in the IL but not in the neighboring regions compared to SHAM. Subsequently, two groups of rats were subjected to fear conditioning, extinction, and retention while receiving stimulation during the extinction. Rats in the tFUS group froze significantly less than SHAM during both extinction and retention tests. Importantly, the reduced freezing in the tFUS group was not attributable to non-specific effect such as auditory noise, as both groups demonstrated a similar level of locomotive activity in an open field regardless of the stimulation condition. Finally, we replicated the procedure with a shortened conditioning-to-extinction interval (15 min) to induce immediate extinction deficit. The tFUS group showed a facilitated reduction in freezing during the extinction, which persisted in the subsequent retention session compared to SHAM. In summary, the current findings suggest that tFUS stimulation in the IL facilitates fear extinction, offering a potential therapeutic regimen for fear-related psychiatric disorders.

Study on the effect of human sympathetic nerve cold stimulation to relieve driving fatigue based on order recurrence plot

Driving fatigue is very likely to cause traffic accidents, seriously threatening the lives and properties of drivers. Therefore, accurate detection and effective mitigation of driving fatigue are crucial for ensuring the personal safety of drivers. This study proposes a method to relieve driving fatigue by properly reducing the temperature to stimulate the human sympathetic nerve. The method uses the intelligent cooling and blowing device on the car seat cushion to achieve cold stimulation of the sympathetic nerve of the driver by reducing the temperature of the driver’s hip, back and neck, so as to increase the excitement of the sympathetic nerve, keep the driver alert and achieve the purpose of fighting driving fatigue. In view of the fact that the traditional fatigue detection method is easily affected by environmental factors and individual differences, this study uses the order recurrence plot (ORP) method to detect driving fatigue based on electroencephalogram (EEG) signals. The results show that ORP textures drawn by EEG signals of the two driving conditions (normal driving condition and sensory cold stimulation driving condition) are significantly different, and the quantization parameters determinism (DET) and average diagonal line length (DLL) values are significantly different. Cold stimulation of the subjects’ hips, back and neck to alleviate driving fatigue was the best when the temperature was 21 °C. In addition, compared with the traditional methods of fatigue relief, the sensory cold stimulation method proposed in this study does not easily to produce tolerance and has no damage to the body.

Assessment of focused ultrasound stimulation to induce peripheral nerve activity and potential damage in vivo.

Peripheral neuroprostheses are aimed to restore loss of sensory and motor functions by interfacing axons in the peripheral nerves. Most common interfaces in neuroprostheses are electrodes that establish electrical connection with peripheral axons. However, some challenges arise related to long-term functionality, durability, and body response. Recently, focused ultrasound stimulation (FUS) has emerged as a non-invasive approach to modulate the nervous system. However, it is controversial whether FUS can induce axon depolarization. We have assessed FUS applied in vivo to the rat peripheral nerve, with two objectives: first, to test whether FUS activates peripheral nerves under different stimulation conditions, and second, to evaluate if FUS inflicts damage to the nerve. FUS was delivered with three ultrasound transducers (Sonic Concept H115, H107, and H102) covering the largest set of parameters examined for FUS of peripheral nerves so far. We did not obtain reliable evoked action potentials in either nerves or muscles, under any FUS condition applied, neither over the skin nor directly to the nerve exposed. Additional experiments ex vivo and in vivo on mice, confirmed this conclusion. When FUS stimulation was applied directly to the exposed sciatic nerve, neuromuscular function decreased significantly, and recovered one week later, except for FUS at 0.25 MHz. Histologically, degenerating nerve fibers were observed, with a tendency to be higher with the lower FUS frequency. Past reports on the ability of ultrasound to stimulate the peripheral nerve are controversial. After testing a wide range of FUS conditions, we conclude that it is not a reliable and safe method for stimulating the peripheral nerve. Special consideration should be taken, especially when low-frequency FUS is applied, as it may lead to nerve damage.

Combining transcranial magnetic stimulation with training to improve social cognition impairment in schizophrenia: a pilot randomized controlled trial.

Schizophrenia is a severe, chronic mental disorder that profoundly impacts patients' everyday lives. The illness's core features include positive and negative symptoms and cognitive impairments. In particular, deficits in the social cognition domain showed a tighter connection to patients' everyday functioning than the other symptoms. Social remediation interventions have been developed, providing heterogeneous results considering the possibility of generalizing the acquired improvements in patients' daily activities. In this pilot randomized controlled trial, we investigated the feasibility of combining fifteen daily cognitive and social training sessions with non-invasive brain stimulation to boost the effectiveness of the two interventions. We delivered intermittent theta burst stimulation (iTBS) over the left dorsolateral prefrontal cortex (DLPFC). Twenty-one patients were randomized into four groups, varying for the assigned stimulation condition (real vs. sham iTBS) and the type of cognitive intervention (training vs. no training). Clinical symptoms and social cognition tests were administered at five time points, i.e., before and after the treatment, and at three follow-ups at one, three, and six months after the treatments' end. Preliminary data show a trend in improving the competence in managing emotion in participants performing the training. Conversely, no differences were found in pre and post-treatment scores for emotion recognition, theory of mind, and attribution of intentions scores. The iTBS intervention did not induce additional effects on individuals' performance. The methodological approach's novelty and limitations of the present study are discussed.

Unraveling the brain mechanisms of source monitoring with non-invasive brain stimulation: A systematic review

Background/ObjectiveSource monitoring refers to the ability to determine the source of memories and encompasses three subprocesses: internal source monitoring, reality monitoring, and external source monitoring. Neuroimaging studies provide valuable insights about neural correlates of source monitoring, but the causal relationship between brain and behavior is lacking. This study aimed to identify brain circuits involved in source monitoring by synthesizing the effects of brain stimulation on source monitoring as a function of the targeted brain regions or circuits. MethodWe conducted a systematic review of interventional studies that have examined the effects of brain stimulation on source monitoring across six databases. The principal outcome was the difference of source monitoring performance between active and control stimulation conditions. Results23 studies (920 healthy participants and 54 patients with schizophrenia) were included. Our findings revealed the involvement of i) the lateral prefrontal and temporoparietal cortices in internal source monitoring, ii) the medial prefrontal and temporoparietal cortices in reality monitoring, and iii) the precuneus and the left angular gyrus in external source monitoring. ConclusionsThese findings deepen our understanding of the brain mechanisms of source monitoring and highlight specific stimulation targets to alleviate source monitoring deficits.

Frontal midline theta transcranial alternating current stimulation enhances early consolidation of episodic memory

Evidence implicating theta rhythms in declarative memory encoding and retrieval, together with the notion that both retrieval and consolidation involve memory reinstatement or replay, suggests that post-learning theta rhythm modulation can promote early consolidation of newly formed memories. Building on earlier work employing theta neurofeedback, we examined whether theta-frequency transcranial alternating stimulation (tACS) can engender effective consolidation of newly formed episodic memories, compared with beta frequency stimulation or sham control conditions. We compared midline frontal and posterior parietal theta stimulation montages and examined whether benefits to memory of theta upregulation are attributable to consolidation rather than to retrieval processes by using a washout period to eliminate tACS after-effects between stimulation and memory assessment. Four groups of participants viewed object pictures followed by a free recall test during three study-test cycles. They then engaged in tACS (frontal theta montage/parietal theta montage/frontal beta montage/sham) for a period of 20 min, followed by a 2-h break. Free recall assessments were conducted after the break, 24 h later, and 7 days later. Frontal midline theta-tACS induced significant off-line retrieval gains at all assessment time points relative to all other conditions. This indicates that theta upregulation provides optimal conditions for the consolidation of episodic memory, independent of mental-state strategies.

Auditory stimulation during REM sleep modulates REM electrophysiology and cognitive performance

REM sleep is critical for memory, emotion, and cognition. Manipulating brain activity during REM could improve our understanding of its function and benefits. Earlier studies have suggested that auditory stimulation in REM might modulate REM time and reduce rapid eye movement density. Building on this, we studied the cognitive effects and electroencephalographic responses related to such stimulation. We used acoustic stimulation locked to eye movements during REM and compared two overnight conditions (stimulation and no-stimulation). We evaluated the impact of this stimulation on REM sleep duration and electrophysiology, as well as two REM-sensitive memory tasks: visual discrimination and mirror tracing. Our results show that this auditory stimulation in REM decreases the rapid eye movements that characterize REM sleep and improves performance on the visual task but is detrimental to the mirror tracing task. We also observed increased beta-band activity and decreased theta-band activity following stimulation. Interestingly, these spectral changes were associated with changes in behavioural performance. These results show that acoustic stimulation can modulate REM sleep and suggest that different memory processes underpin its divergent impacts on cognitive performance.

Soleus H-reflex amplitude modulation during walking remains physiological during transspinal stimulation in humans.

The soleus H-reflex modulation pattern was investigated during stepping following transspinal stimulation over the thoracolumbar region at 15, 30, and 50Hz with 10kHz carry-over frequency above and below the paresthesia threshold. The soleus H-reflex was elicited by posterior tibial nerve stimulation with a single 1 ms pulse at an intensity that the M-wave amplitudes ranged from 0 to 15% of the maximal M-wave evoked 80ms after the test stimulus, and the soleus H-reflex was half the size of the maximal H-reflex evoked on the ascending portion of the recruitment curve. During treadmill walking, the soleus H-reflex was elicited every 2 or 3 steps, and stimuli were randomly dispersed across the step cycle which was divided in 16 equal bins. For each subject and condition, the soleus M-wave and H-reflex were normalized to the maximal M-wave. The soleus background electromyographic (EMG) activity was estimated as the linear envelope for 50ms duration starting at 100ms before posterior tibial nerve stimulation for each bin. The gain was determined as the slope of the relationship between the soleus H-reflex and the soleus background EMG activity. The soleus H-reflex phase-dependent amplitude modulation remained unaltered during transspinal stimulation, regardless frequency, or intensity. Similarly, the H-reflex slope and intercept remained the same for all transspinal stimulation conditions tested. Locomotor EMG activity was increased in knee extensor muscles during transspinal stimulation at 30 and 50Hz throughout the step cycle while no effects were observed in flexor muscles. These findings suggest that transspinal stimulation above and below the paresthesia threshold at 15, 30, and 50Hz does not block or impair spinal integration of proprioceptive inputs and increases activity of thigh muscles that affect both hip and knee joint movement. Transspinal stimulation may serve as a neurorecovery strategy to augment standing or walking ability in upper motoneuron lesions.

Demonstration of multi-point stimulation with AC-driven CMOS chips for retinal prosthesis

We demonstrated a multi-point stimulation system using CMOS chips designed for distributed implantable retinal prostheses. The CMOS chip integrates current sources and electrode selection circuits. This integration enables stimulation current control from multiple electrodes with minimal wires. In this study, we constructed a validation system with 49 electrodes capable of visually observing stimulation current outputs. The validation results show that it is possible to configure stimulation conditions in approximately 9.3 μs per electrode, and we demonstrated the ability to complete one frame, including the stimulation time, within approximately 11 ms. We also demonstrated parallel current stimulation from multiple electrodes using an in vitro experiment.

Sleepy without stimulation: subjective and objective sleepiness in actigraphy-verified natural short sleepers.

Natural short sleepers (NSS)-individuals who report minimal sleepiness or daytime dysfunction despite habitually sleeping less than the recommended amount (i.e., <7 h)-are a focus of growing interest in sleep research. Yet, the predominance of research on NSS has relied on subjective reports of functionality. The present study examined subjective and objective sleepiness among actigraphy-verified NSS in comparison with recommended (7-9 h/day) length sleepers (RLS) who reported similarly minimal daytime dysfunction. The study tested the hypothesis that under conditions of low environmental stimulation, NSS have increased risk of drowsiness and sleep onset, regardless of perceived alertness. The NSS and RLS groups were identified via screening and verified with a 14 day assessment with actigraphy, sleep diaries, and morning ratings of sleep restoration. In-laboratory resting electroencephalography (EEG) data were analysed using a computerised EEG-based algorithm (Vigilance Algorithm Leipzig; VIGALL) to classify second-by-second changes in objective sleepiness ranging from cognitively active alertness to sleep onset. Results demonstrated that NSS exhibited significantly higher drowsiness and sleep onset ('microsleeps') across 15 min of resting EEG despite perceptions of lower subjective sleepiness compared to RLS. Findings suggest that irrespective of perceived sleep restoration and alertness, NSS appear to be at high risk of objective sleepiness that is rapidly unmasked under conditions of low environmental stimulation. Such apparent discrepancy between subjective and objective sleepiness has potentially important public health implications. Future research directions, including tests of mechanisms and tailored sleep extension intervention, are discussed.

Transcutaneous auricular vagus nerve stimulation alters cough sensitivity depending on stimulation parameters: potential implications for aspiration risk.

Transcutaneous auricular vagus nerve stimulation (taVNS) is considered a safe and promising tool for limb rehabilitation after stroke, but its effect on cough has never been studied. It is known that the ear and larynx share vagal afferent pathways, suggesting that stimulating the ear with taVNS might have effects on cough sensitivity. The specific stimulation parameters used can influence outcomes. To investigate the effect of various stimulation parameters on change in cough sensitivity, compared to the reference parameter of 25 Hz stimulation at the left concha (most commonly-used parameter for stroke rehabilitation). Design, setting, and participants: Randomized, single-blind, active-controlled, eight-period cross-over design conducted March to August 2022 at a New Zealand research laboratory with 16 healthy participants. All participants underwent eight stimulation conditions which varied by stimulation side (right ear, left ear), zone (ear canal, concha), and frequency (25 Hz, 80 Hz). Main outcome measures: Change in natural and suppressed cough threshold (from baseline to after 10 min of stimulation) assessed using a citric acid cough reflex test. When compared to the reference parameter of 25 Hz stimulation at the left concha, there was a reduction in natural cough threshold of -0.16 mol/L for 80 Hz stimulation at the left canal (p = 0.004), indicating increased sensitivity. For the outcome measure of suppressed cough threshold, there was no significant effect of any of the stimulation conditions compared to the active reference. Since stroke patients often have cough hyposensitivity with resulting high risk of silent aspiration, using 80 Hz taVNS at the left canal may be a better choice for future stroke rehabilitation studies than the commonly used 25 Hz taVNS at the left concha. Treatment parameters should be manipulated in future sham-controlled trials to maximize any potential treatment effect of taVNS in modulating cough sensitivity. ACTRN12623000128695.

Slow waves during deep sleep support cardiac function

Introduction The role of sleep in cardiovascular health, particularly the impact of deep non-rapid eye movement (NREM) sleep, is gaining interest in the prevention of cardiovascular diseases (Grandner et al., 2016). Essentially slow waves, prominent brain oscillations during deep sleep, seem to represent an important cardiovascular recovery process for the human body. However, to date, the specific mechanisms through which sleep affects cardiovascular function and whether slow waves accelerate recovery remain elusive. Methods Here, we explored the role of slow waves in promoting cardiovascular function. In this randomized, cross-over trial involving 18 healthy male participants (Huwiler et al., 2023), we investigated how experimentally enhancing slow waves via auditory stimulation (Huwiler et al., 2022) impacts cardiovascular dynamics during sleep and next-day cardiac function, assessed via an echocardiography. All participants underwent three experimental nights including two conditions for slow wave enhancement and one SHAM control condition while brain oscillatory, blood pressure, and cardiac dynamics were continuously measured during sleep. Results Using a linear mixed-effect model approach, we found auditory stimulation to significantly increase slow wave activity (F(2, 33.095) = 11.397, p &lt; 0.001) during times of stimulation. This slow wave enhancement coincided with a dynamic cardiovascular activation, indicated through short increases in blood pressure and a biphasic heart rate response. After sleep, we found a significant increase in general longitudinal strain (F(2, 34) = 81.17, p &lt; 0.001), an increase in left-ventricular ejection fraction (F(2, 34) = 4.55, p = 0.018), and a decrease in E/e’ ratio (F(2, 34) = 3.38, p = 0.046) for both slow wave stimulation conditions compared to SHAM. Discussion/Conclusion We show that slow wave stimulation induces a cardiovascular activation response possibly related to increasing cardiovascular stability during sleep (De Zambotti et al., 2016). Moreover, we demonstrate that slow wave stimulation enhances left-ventricular systolic and diastolic function, both together indicating improved cardiac function. Altogether, this points towards the functional involvement of slow waves in promoting cardiovascular health. Therefore, sleep slow wave stimulation may be considered a potential supplementary method in treating cardiovascular diseases involving decreased left-ventricular function. Furthermore, our findings hint that enhancing slow waves could potentially optimize post-exercise recovery processes and increase cardiovascular well-being.

Neural activity of retinal ganglion cells under continuous, dynamically-modulated high frequency electrical stimulation

Objective. Current retinal prosthetics are limited in their ability to precisely control firing patterns of functionally distinct retinal ganglion cell (RGC) types. The aim of this study was to characterise RGC responses to continuous, kilohertz-frequency-varying stimulation to assess its utility in controlling RGC activity. Approach. We used in vitro patch-clamp experiments to assess electrically-evoked ON and OFF RGC responses to frequency-varying pulse train sequences. In each sequence, the stimulation amplitude was kept constant while the stimulation frequency (0.5–10 kHz) was changed every 40 ms, in either a linearly increasing, linearly decreasing or randomised manner. The stimulation amplitude across sequences was increased from 10 to 300 µA. Main results. We found that continuous stimulation without rest periods caused complex and irreproducible stimulus-response relationships, primarily due to strong stimulus-induced response adaptation and influence of the preceding stimulus frequency on the response to a subsequent stimulus. In addition, ON and OFF populations showed different sensitivities to continuous, frequency-varying pulse trains, with OFF cells generally exhibiting more dependency on frequency changes within a sequence. Finally, the ability to maintain spiking behaviour to continuous stimulation in RGCs significantly reduced over longer stimulation durations irrespective of the frequency order. Significance. This study represents an important step in advancing and understanding the utility of continuous frequency modulation in controlling functionally distinct RGCs. Our results indicate that continuous, kHz-frequency-varying stimulation sequences provide very limited control of RGC firing patterns due to inter-dependency between adjacent frequencies and generally, different RGC types do not display different frequency preferences under such stimulation conditions. For future stimulation strategies using kHz frequencies, careful consideration must be given to design appropriate pauses in stimulation, stimulation frequency order and the length of continuous stimulation duration.

Effects of noninvasive neuromodulation targeting the spinal cord on early learning of force control by the digits.

Control of finger forces underlies our capacity for skilled hand movements acquired during development and reacquired after neurological injury. Learning force control by the digits, therefore, predicates our functional independence. Noninvasive neuromodulation targeting synapses that link corticospinal neurons onto the final common pathway via spike-timing-dependent mechanisms can alter distal limb motor output on a transient basis, yet these effects appear subject to individual differences. Here, we investigated how this form of noninvasive neuromodulation interacts with task repetition to influence early learning of force control during precision grip. The unique effects of neuromodulation, task repetition, and neuromodulation coinciding with task repetition were tested in three separate conditions using a within-subject, cross-over design (n = 23). We found that synchronizing depolarization events within milliseconds of stabilizing precision grip accelerated learning but only after accounting for individual differences through inclusion of subjects who showed upregulated corticospinal excitability at 2 of 3 time points following conditioning stimulation (n = 19). Our findings provide insights into how the state of the corticospinal system can be leveraged to drive early motor skill learning, further emphasizing individual differences in the response to noninvasive neuromodulation. We interpret these findings in the context of biological mechanisms underlying the observed effects and implications for emerging therapeutic applications.

Dual electrical stimulation at spinal-muscular interface reconstructs spinal sensorimotor circuits after spinal cord injury

The neural signals produced by varying electrical stimulation parameters lead to characteristic neural circuit responses. However, the characteristics of neural circuits reconstructed by electrical signals remain poorly understood, which greatly limits the application of such electrical neuromodulation techniques for the treatment of spinal cord injury. Here, we develop a dual electrical stimulation system that combines epidural electrical and muscle stimulation to mimic feedforward and feedback electrical signals in spinal sensorimotor circuits. We demonstrate that a stimulus frequency of 10−20 Hz under dual stimulation conditions is required for structural and functional reconstruction of spinal sensorimotor circuits, which not only activates genes associated with axonal regeneration of motoneurons, but also improves the excitability of spinal neurons. Overall, the results provide insights into neural signal decoding during spinal sensorimotor circuit reconstruction, suggesting that the combination of epidural electrical and muscle stimulation is a promising method for the treatment of spinal cord injury.

Impacts of Deep Brain Stimulation of the Globus Pallidus Internus on Swallowing: A Retrospective, Cross-Sectional Study.

Deep brain stimulation (DBS) is a common treatment for motor symptoms of Parkinson disease (PD), a condition associated with increased risk of dysphagia. The effect of DBS on swallowing function has not been comprehensively evaluated using gold-standard imaging techniques, particularly for globus pallidus internus (GPi) DBS. The objective of this retrospective, cross-sectional study was to identify differences in swallowing safety and timing kinematics among PD subjects with and without GPi DBS. We investigated the effects of unilateral and bilateral GPi DBS as well as the relationship between swallowing safety and DBS stimulation parameters, using retrospective analysis of videofluoroscopy recordings (71 recordings from 36 subjects) from electronic medical records. Outcomes were analyzed by surgical status (pre-surgical, unilateral DBS, bilateral DBS). The primary outcome was percent of thin-liquid bolus trials rated as unsafe, with Penetration-Aspiration Scale scores of 3 or higher. Secondary analyses included swallowing timing measures, relationships between swallowing safety and DBS stimulation parameters, and Dynamic Imaging Grade of Swallowing Toxicity ratings. Most subjects swallowed all boluses safely (19/29 in the pre-surgical, 16/26 in the unilateral DBS, and 10/16 in the bilateral DBS conditions). Swallowing safety impairment did not differ among stimulation groups. There was no main effect of stimulation condition on timing metrics, though main effects were found for sex and bolus type. Stimulation parameters were not correlated with swallowing safety. Swallowing efficiency and overall impairment did not differ among conditions. These results provide evidence that GPi DBS does not affect pharyngeal swallowing function. Further, prospective, investigations are needed.

OTUD3 suppresses the mTORC1 signaling by deubiquitinating KPTN.

Background: Ubiquitination and deubiquitination modifications play pivotal roles in eukaryotic life processes, regulating protein dynamics via the ubiquitin-proteasome pathway. Dysregulation can impact disease development, including cancer and neurodegenerative disorders. Increasing evidence highlights their role in tumorigenesis, modulating key proteins. OTUD3, a deubiquitinase, stabilizes PTEN, suppressing tumor growth by inhibiting PI3K-AKT signaling. Yet, further OTUD3 substrates remain underexplored. Methods: We employed the In vivo ubiquitination assay to investigate the ubiquitination role of OTUD3 on KPTN within the cellular context. Additionally, CRISPR/Cas9 editing and Immunofluorescence were utilized to study the impact of OTUD3 on the mTOR signaling pathway in cells. Furthermore, Cell proliferation assay and NMR were employed to explore the effects of OTUD3 on cellular growth and proliferation. Results: OTUD3 serves as a deubiquitinase for KPTN. OTUD3 interacts with KPTN, facilitated by the OTU domain within OTUD3. Further investigations confirmed KPTN's ubiquitination modification, primarily at lysine residue 49. Ubiquitination experiments demonstrated OTUD3's ability to mediate KPTN's deubiquitination without affecting its protein levels. This suggests KPTN's ubiquitination is a function-regulated, non-degradable modification. Under various amino acid starvation or stimulation conditions, overexpressing OTUD3 reduces mTORC1 signaling activation, while knocking out OTUD3 further enhances it. Notably, OTUD3's regulation of mTORC1 signaling relies on its deubiquitinase activity, and this effect is observed even in PTEN KO cells, confirming its independence from PTEN, a reported substrate. OTUD3 also promotes GATOR1's lysosomal localization, a process requiring KPTN's involvement. Ultimately, OTUD3 affects cellular metabolic pool products by downregulating the mTORC1 pathway, significantly inhibiting tumor cell growth and proliferation. Discussion: Our experiments shed light on an alternative perspective regarding the intrinsic functions of OTUD3 in inhibiting tumor development. We propose a novel mechanism involving KPTN-mediated regulation of the mTORC1 signaling pathway, offering fresh insights into the occurrence and progression of tumor diseases driven by related genes. This may inspire new approaches for drug screening and cancer treatment, potentially guiding future therapies for relevant tumors.

A novel mass cytometry protocol optimized for immunophenotyping of low-frequency antigen-specific T cells.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2-vaccinated and SARS-CoV-2-infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.

Pro-Inflammatory Cytokines Enhanced In Vitro Cytotoxic Activity of Clostridioides difficile Toxin B in Enteric Glial Cells: The Achilles Heel of Clostridioides difficile Infection?

Bacterial infections are characterized by an inflammatory response, which is essential for infection containment but is also responsible for negative effects on the host. The pathogen itself may have evolved molecular mechanisms to antagonize the antimicrobial effects of an inflammatory response and to enhance its pathogenicity using inflammatory response mediators, such as cytokines. Clostridioides difficile (C. difficile) infection (CDI) causes gastrointestinal diseases with markedly increasing global incidence and mortality rates. The main C. difficile virulence factors, toxin A and B (TcdA/TcdB), cause cytopathic/cytotoxic effects and inflammation. We previously demonstrated that TcdB induces enteric glial cell (EGC) apoptosis, which is enhanced by the pro-inflammatory cytokine tumor necrosis factor alpha plus interferon gamma (CKs). However, it is unknown whether CKs-enhanced TcdB cytotoxicity (apoptosis/necrosis) is affected by the timing of the appearance of the CKs. Thus, we simulated in vitro, in our experimental model with TcdB and EGCs, three main situations of possible interactions between TcdB and the timing of CK stimulation: before TcdB infection, concomitantly with infection, or at different times after infection and persisting over time. In these experimental conditions, which all represent situations of possible interactions between C. difficile and the timing of CK stimulation, we evaluated apoptosis, necrosis, and cell cycle phases. The CKs, in all of these conditions, enhanced TcdB cytotoxicity, which from apoptosis became necrosis when CK stimulation persisted over time, and was most relevant after 48 h of TcdB:EGCs interaction. Particularly, the enhancement of apoptosis by CKs was dependent on the TcdB dose and in a less relevant manner on the CK stimulation time, while the enhancement of necrosis occurred always independently of the TcdB dose and CK stimulation time. However, since in all conditions stimulation with CKs strongly enhanced the TcdB cytotoxicity, it always had a negative impact on C. difficile pathogenicity. This study might have important implications for the treatment of CDI.