Single-cell omics technologies, such as single-cell RNA-Seq and single-cell proteomics, offer unprecedented insights into cellular heterogeneity and dynamic regulatory processes. However, integrating these data types to construct comprehensive transcription-translation profiles remains challenging because of their distinct and complex behaviors. This study presents a novel approach using pseudotemporal cell ordering to integrate single-cell RNA-Seq and single-cell proteomics by mass spectrometry data, facilitating the analysis of transcription-translation expression dynamics. We collected longitudinal single-cell samples following hypoxia. By leveraging key markers, we constructed pseudotemporal trajectories for each data type, revealing transcriptional and translational responses to hypoxia. This profile of unified single-cell mRNA and protein expression uncovers distinct regulatory mechanisms, including an immediate transcriptomic response, followed by delayed proteomic expression. It illustrates the use of pseudotemporal integration to integrate single-cell transcriptomic and proteomic datasets to understand the cellular phenotypes under hypoxic stress and provides a framework for future investigations into transcription-translation dynamics.

Use of one addictive drug typically influences the behavioral response to other drugs, either administered at the same time or a subsequent time point. The nature of the drugs being used, as well as the timing and dosing, also influence how these drugs interact. Here, we tested the effects of adolescent THC exposure on the development of morphine-induced behavioral adaptations following repeated morphine exposure during adulthood. We found that adolescent THC administration paradoxically prevented the development of anxiety-related behaviors that emerge during a forced abstinence period following morphine administration but facilitated reinstatement of morphine CPP. Following forced abstinence, we then mapped the whole-brain response to a moderate dose of morphine and found that adolescent THC administration led to an overall increase in brain-wide neuronal activity and increased the functional connectivity between frontal cortical regions and the ventral tegmental area. Last, we show using rabies virus-based circuit mapping that adolescent THC exposure triggers a long-lasting elevation in connectivity from the frontal cortex regions onto ventral tegmental dopamine cells. Our study adds to the rich literature on the interaction between drugs, including THC and opioids, and provides potential neural substates by which adolescent THC exposure influences responses to morphine later in life.

Rationale Adolescent cannabis use is linked to later-life changes in cognition, learning, and memory. Rodent experimental studies suggest Δ9-tetrahydrocannabinol (THC) influences development of circuits underlying these processes, especially in the prefrontal cortex, which matures during adolescence. Objective We determined how 14 daily THC injections (5mg/kg) during adolescence persistently impacts medial prefrontal cortex (mPFC) dopamine-dependent cognition. Methods In adult Long Evans rats treated as adolescents with THC (AdoTHC), we quantify performance on two mPFC dopamine-dependent reward-based tasks—strategy set shifting and probabilistic discounting. We also determined how acute dopamine augmentation with amphetamine (0, 0.25, 0.5 mg/kg), or specific chemogenetic stimulation of ventral tegmental area (VTA) dopamine neurons and their projections to mPFC impacts probabilistic discounting. Results AdoTHC sex-dependently impacts acquisition of cue-guided instrumental reward seeking, but has minimal effects on set-shifting or probabilistic discounting in either sex. When we challenged dopamine circuits acutely with amphetamine during probabilistic discounting, we found reduced discounting of improbable reward options, with AdoTHC rats being more sensitive to these effects than controls. In contrast, neither acute chemogenetic stimulation of VTA dopamine neurons nor pathway-specific chemogenetic stimulation of their projection to mPFC impacted probabilistic discounting in control rats, although stimulation of this cortical dopamine projection slightly disrupted choices in AdoTHC rats. Conclusions These studies confirm a marked specificity in the cognitive processes impacted by AdoTHC exposure. They also suggest that some persistent AdoTHC effects may alter amphetamine-induced cognitive changes in a manner independent of VTA dopamine neurons or their projections to mPFC.

Chinese hamster ovary (CHO) cells are widely used to produce complex biopharmaceuticals. Improving their productivity is necessary to fulfill the growing demand for such products. One way to enhance productivity is by cultivating cells at high densities, but inhibitory by-products, such as metabolite derivatives from amino acid degradation, can hinder achieving high cell densities. This research examines the impact of these inhibitory by-products on high-density cultures. We cultured X1 and X2 CHO cell lines in a small-scale semi-perfusion system and introduced a mix of inhibitory by-products on day 10. The X1 and X2 cell lines were chosen for their varied responses to the by-products; X2 was susceptible, while X1 survived. Proteomics revealed that the X2 cell line presented changes in the proteins linked to apoptosis regulation, cell building block synthesis, cell growth, DNA repair, and energy metabolism. We later used the AB cell line, an apoptosis-resistant cell line, to validate the results. AB behaved similar to X1 under stress. We confirmed the activation of apoptosis in X2 using a caspase assay. This research provides insights into the mechanisms of cell death triggered by inhibitory by-products and can guide the optimization of CHO cell culture for biopharmaceutical manufacturing.

Opioid use disorder (OUD) is associated with a history of early-life adversity (ELA), an association that is particularly strong in women. In a rodent model, we previously found that ELA enhances risk for opioid addiction selectively in females, but the mechanisms for this effect are unclear. Here, we show that ELA robustly alters cFos responses to opioid drugs in females’ nucleus accumbens (NAc) and basolateral amygdala (BLA), but not elsewhere. We further identify delta opioid receptors (DOR), which mature in the first week of life and thus later than kappa or mu opioid receptors, as a potential mediator of ELA's impacts on reward circuit functions. Accordingly, DOR mRNA in NAc was persistently reduced in adult females with ELA history. Moreover, pharmacological stimulation of NAc DORs increased opioid demand in control females (recapitulating the ELA phenotype), while blocking DORs in ELA females conversely reduced high-effort drug consumption, simulating the control rearing phenotype. These findings support a role for NAc DORs in mediating ELA-induced opioid vulnerability. In contrast, BLA neurons expressing DOR protein do not overlap heroin- responsive cells in ELA rats, arguing against a direct relationship of BLA DORs to heroin's addiction-relevant actions in the brain. Together, these results suggest a novel and selective role for NAc DORs in contributing to enduring, ELA-provoked vulnerability to OUD.

Introduction: Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are major chemical constituents of cannabis, which may interact either directly or indirectly with the endocannabinoid and endocannabinoid-like ("paracannabinoid") systems, two lipid-based signaling complexes that play important roles in physiology. Legislative changes emphasize the need to understand how THC and CBD might impact endocannabinoid and paracannabinoid signaling, and to develop analytical approaches to study such impact. In this study, we describe a sensitive and accurate method for the simultaneous quantification of THC, its main oxidative metabolites [11-hydroxy-Δ9-THC (11-OH-THC) and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC)], CBD, and a representative set of endocannabinoid [anandamide and 2-arachidonoyl-sn-glycerol (2-AG)] and paracannabinoid [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)] compounds. Analyte separation relies on the temperature-dependent shape selectivity properties of polymerically bonded C18 stationary phases. Materials and Methods: Analytes are extracted from tissues using acetonitrile precipitation followed by phospholipid removal. The ultrahigh-performance liquid chromatography/tandem mass spectrometry protocol utilizes a commercially available C18 polymeric-bonded phase column and a simple gradient elution system. Results: Ten-point calibration curves show excellent linearity (R2>0.99) over a wide range of analyte concentrations (0.02-500 ng/mL). Lowest limits of quantification are 0.05 ng/mL for anandamide, 0.1 ng/mL for 11-OH-THC and OEA, 0.2 ng/mL for THC and CBD, 0.5 ng/mL for 11-COOH-THC, 1.0 ng/mL for 2-AG, and 2.0 ng/mL for PEA. The lowest limits of detection are 0.02 ng/mL for anandamide, 0.05 ng/mL for 11-OH-THC and OEA, 0.1 ng/mL for THC and CBD, 0.2 ng/mL for 11-COOH-THC, 0.5 ng/mL for 2-AG, and 1.0 ng/mL for PEA. Conclusions: An application of the method is presented, which showed that phytocannabinoid administration elevates endocannabinoid levels in plasma and brain of adolescent male and female mice.

Rationale The endocannabinoid system makes critical contributions to reward processing, motivation, and behavioral control. Repeated exposure to THC or other cannabinoid drugs can cause persistent adaptions in the endocannabinoid system and associated neural circuitry. It remains unclear how such treatments affect the way rewards are processed and pursued. Objective and methods We examined if repeated THC exposure (5 mg/kg/day for 14 days) during adolescence or adulthood led to long-term changes in rats’ capacity to flexibly encode and use action-outcome associations for goal-directed decision making. Effects on hedonic feeding and progressive ratio responding were also assessed. Results THC exposure had no effect on rats’ ability to flexibly select actions following reward devaluation. However, instrumental contingency degradation learning, which involves avoiding an action that is unnecessary for reward delivery, was augmented in rats with a history of adult but not adolescent THC exposure. THC-exposed rats also displayed more vigorous instrumental behavior in this study, suggesting a motivational enhancement. A separate experiment found that while THC exposure had no effect on hedonic feeding behavior, it increased rats’ willingness to work for food on a progressive ratio schedule, an effect that was more pronounced when THC was administered to adults. Adolescent and adult THC exposure had opposing effects on the CB1 receptor dependence of progressive ratio performance, decreasing and increasing sensitivity to rimonabant-induced behavioral suppression, respectively. Conclusions Our findings reveal that exposure to a translationally relevant THC exposure regimen induces long-lasting, age-dependent alterations in cognitive and motivational processes that regulate the pursuit of rewards.

"Reagentless" immunosensors are emerging to address the challenge of practical and sensitive detection of important biomarkers in real biological samples without the need for multistep assays and user intervention, with applications ranging from research tools to point-of-care diagnostics. Selective target binding to an affinity reagent is detected and reported in one step without the need for washing or additional reporters. In this study, we used a structure-guided approach to identify a mutation site in an antibody fragment for the polarity-dependent fluorophore, Anap, such that upon binding of the protein target cardiac troponin I, the Anap-labeled antibody would produce a detectable and dose-dependent shift in emission wavelength. We observed a significant emission wavelength shift of the Anap-labeled anti-cTnI mutant, with a blue shift of up to 37 nm, upon binding to the cTnI protein. Key differences in the resulting emission spectra between target peptides in comparison to whole proteins were also found; however, the affinity and binding characteristics remained unaffected when compared to the wild-type antibody. We also highlighted the potential flexibility of the approach by incorporating a near-infrared dye, IRDye800CW, into the same mutation site, which also resulted in a dose-dependent wavelength shift upon target incubation. These reagents can be used in experiments and devices to create simpler and more efficient biosensors across a range of research, medical laboratory, and point-of-care platforms.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

Designer receptors exclusively activated by designer drugs (DREADDs) are a tool for “remote control” of defined neuronal populations during behavior. These receptors are inert unless bound by an experimenter-administered designer drug, commonly clozapine-n-oxide (CNO). However, questions have emerged about the suitability of CNO as a systemically administered DREADD agonist. Second-generation agonists such as JHU37160 (J60) have been developed, which may have more favorable properties than CNO. Here we sought to directly compare effects of CNO (0, 1, 5, 10 mg/kg, i.p.) and J60 (0, 0.03, 0.3, 3 mg/kg, i.p.) on operant food pursuit. Male and female TH:Cre + rats and their wildtype (WT) littermates received cre-dependent hM4Di-mCherry vector injections into ventral tegmental area (VTA), causing inhibitory DREADD expression in VTA dopamine neurons of TH:Cre + rats. All rats were trained to stably lever press for palatable food on a fixed ratio 10 schedule, and doses of both agonists were tested on separate days in counterbalanced order. All three CNO doses reduced operant rewards earned in rats with DREADDs, and no CNO dose had behavioral effects in WT controls. The highest J60 dose tested significantly reduced responding in DREADD rats, but this dose also increased responding in WTs, indicating non-specific effects. The magnitude of CNO and J60 effects in TH:Cre + rats were correlated and were present in both sexes. Findings demonstrate the usefulness of directly comparing DREADD agonists when optimizing behavioral chemogenetics, and highlight the importance of proper controls, regardless of the DREADD agonist employed.

Passive aerosol exposure to A9-tetrahydrocannabinol (THC) in laboratory animals results in faster onset of action and less extensive liver metabolism compared to most other administration routes and might thus provide an ecologically relevant model of human cannabis inhalation. Previous studies have, however, overlooked the possibility that rodents, as obligate nose breathers, may accumulate aerosolized THC in the nasal cavity, from where the drug might directly diffuse to the brain. To test this, we administered THC (ten 5-s puffs of 100 mg/mL of THC) to adolescent (31-day-old) Sprague-Dawley rats of both sexes. We used liquid chromatography/tandem mass spectrometry to quantify the drug and its first-pass metabolites - 11-hydroxy-A9-THC (11-OH-THC) and 11nor-9-carboxy-A9-THC (11-COOH-THC) - in nasal mucosa, lungs, plasma, and brain (olfactory bulb and cerebellum) at various time points after exposure. Apparent maximal THC concentration and area under the curve were -5 times higher in nasal mucosa than in lungs and 50-80 times higher than in plasma. Concentrations of 11-OH-THC were also greater in nasal mucosa and lungs than other tissues, whereas 11-COOH-THC was consistently undetectable. Experiments with microsomal preparations confirmed local metabolism of THC into 11-OH-THC (not 11-COOH-THC) in nasal mucosa and lungs. Finally, whole-body exposure to THC deposited substantial amounts of THC (-150 mg/g) on fur but suppressed post-exposure grooming in rats of both sexes. The results indicate that THC absorption and metabolism in nasal mucosa and lungs, but probably not gastrointestinal tract, contribute to the pharmacological effects of aerosolized THC in male and female rats.

Disrupted operations of the reward circuit underlie major emotional disorders, including depression, which commonly arise following early life stress / adversity (ELA). However, how ELA enduringly impacts reward circuit functions remains unclear. We characterize a stress-sensitive projection connecting basolateral amygdala (BLA) and nucleus accumbens (NAc) that co-expresses GABA and the stress-reactive neuropeptide corticotropin-releasing hormone (CRH). We identify a crucial role for this projection in executing disrupted reward behaviors provoked by ELA: chemogenetic and optogenetic stimulation of the projection in control male mice suppresses several reward behaviors, recapitulating deficits resulting from ELA and demonstrating the pathway’s contributions to normal reward behaviors. In adult ELA mice, inhibiting–but not stimulating–the projection, restores typical reward behaviors yet has little effect in controls, indicating ELA-induced maladaptive plasticity of this reward-circuit component. Thus, we discover a stress-sensitive, reward inhibiting BLA → NAc projection with unique molecular features, which may provide intervention targets for disabling mental illnesses.

Disrupted operations of the reward circuit are thought to underlie major emotional disorders including depression and drug abuse 1–3 . These disorders commonly arise following early life stress 4,5 ; however, how stress early in life enduringly impacts reward circuit functions to promote disease remains unclear. Here, we discover and characterize a novel stress-sensitive reward-circuit projection connecting the basolateral amygdala (BLA) and nucleus accumbens (NAc) that co-expresses GABA and the stress-reactive neuropeptide corticotropin-releasing hormone (CRH). We then identify a crucial role for this projection in executing the disrupted reward behaviors provoked by early-life adversity (ELA): Chemogenetic and optogenetic stimulations of the CRH GABA BLA→NAc projection in typically reared mice suppressed several reward seeking behaviors, recapitulating deficits resulting from ELA and demonstrating a key contribution of this pathway in the normal operations of the reward circuit. Next, inhibition of the CRH GABA BLA→NAc projection in adult mice that experienced ELA restored typical reward behaviors in these mice, and, in contrast, had little effect in typically reared mice, indicating a selective ELA-induced maladaptive plasticity of this reward-circuit projection. We discover a novel, stress-sensitive, reward inhibiting projection from the BLA→NAc with unique molecular features, which may provide targets for intervention in disabling mental illnesses.

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

Personalised nanomedicine is an advancing field which has developed significant improvements for targeting therapeutics to aggressive cancer and with fewer side effects. The treatment of gliomas such as glioblastoma (or other brain tumours), with nanomedicine is complicated by a commonly poor accumulation of drugs in tumour tissue owing to the partially intact blood-brain barrier (BBB). Nonetheless, the BBB becomes compromised following surgical intervention, and gradually with disease progression. Increased vasculature permeability generated by a tumour, combined with decreased BBB integrity, offers a mechanism to enhance therapeutic outcomes. We monitored a spontaneous glioma tumour model in immunocompetent mice with ongoing T2-weighted and contrast-enhanced T1-weighted magnetic resonance imaging gradient echo and spin echo sequences to predict an optimal "leakiness" stage for nanomedicine injections. To ascertain the effectiveness of targeted nanomedicines in treating brain tumours, subsequent systemic administration of targeted hyperbranched polymers was then utislised, to deliver the therapeutic payload when both the tumour and brain vascularity had become sufficiently susceptible to allow drug accumulation. Treatment with either doxorubicin-loaded hyperbranched polymer, or the same nanomedicine targeted to an ephrin receptor (EphA2) using a bispecific antibody, resulted in uptake of chemotherapeutic doxorubicin in the tumour and in reduced tumour growth. Compared to vehicle and doxorubicin only, nanoparticle delivered doxorubicin resulted in increased tumour apoptosis, while averting cardiotoxicity. This suggests that polyethylene based (PEGylated)-nanoparticle delivered doxorubicin could provide a more efficient treatment in tumours with a disrupted BBB, and that treatment should commence immediately following detection of gadolinium permeability, with early detection and ongoing 'leakiness' monitoring in susceptible patients being a key factor.

Developing new approaches to monitor critical analytes in complex biological environments is key to the development of new diagnostic tools for application in a range of fields including the biomedical, environmental and food sciences. While reversible ion and metabolite sensing can be achieved through a variety of approaches including ionophore-based techniques, there is a lack of approaches to achieve real-time protein monitoring. Immunoassays (e.g. ELISA) are robust approaches to achieve endpoint measurements, however the multi-step nature of the technique is not amenable to reversible biosensing. One approach to simplify immunoassay chemistry is to reduce the number of assay steps required, which could lead to simpler manual assays, less reliance on laboratory infrastructure, or simpler POC designs. We have developed a biosensor approach based on direct detection of antigen-antibody binding in a single step. Using a site-specific approach to incorporate fluorescent dyes into antibody fragments, we screened a range of positional mutants and identified several that showed antigen-dependent spectral changes. These changes were observed to be dose-dependent in both buffer and diluted plasma samples. We then applied a site-directed mutagenesis approach to generate antibody fragments capable of reversible protein interactions and reversible sensing. These fragments can be used in homogenous assays or immobilised to surfaces, and in this presentation we will present our most recent results on epidermal growth factor receptor (EGFR) and cardiac troponin I (cTnI) monitoring.

In humans, early-life adversity (ELA) such as trauma, poverty, and chaotic environment is linked to increased risk of later-life emotional disorders including depression and substance abuse. These disorders involve underlying disruption of reward circuits and likely vary by sex. Accordingly, we previously found that ELA leads to anhedonia for natural rewards and cocaine in male rodents, whereas in females ELA instead increases vulnerability to addiction-like use of opioid drugs and palatable food. While these findings suggest that ELA-induced disruption of reward circuitry may differ between the sexes, the specific circuit nodes that are influenced by ELA in either sex remain poorly understood. Here, in adult male Sprague-Dawley rats, we ask how ELA impacts opioid addiction-relevant behaviors that we previously tested after ELA in females. We probe potential circuit mechanisms in males by assessing opioid-associated neuronal activation in stress and reward circuit nodes including nucleus accumbens (NAc), amygdala, medial prefrontal cortex (mPFC), and paraventricular thalamus. We find that ELA diminishes opioid-seeking behaviors in males, and alters heroin-induced activation of NAc, PFC, and amygdala, suggesting a potential circuit-based mechanism. These studies demonstrate that ELA leads to behavioral and neurobiological disruptions consistent with anhedonia in male rodents, unlike the increased opioid seeking we previously saw in females. Our findings, taken together with our prior work, suggest that men and women could face qualitatively different mental health consequences of ELA, which may be essential for individually tailoring future intervention strategies.