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The oncolytic vesicular stomatitis (VSV)-GP virus is a promising therapeutic against cancer. To ensure clinical efficacy, doses with high titers are required, which poses a challenge for the manufacturing process. Perfusion cultivation processes with high cell densities have attracted great interest to improve the production titer. This work aimed to enhance the titer of the VSV-GP production process with suspension human embryonic kidney 293 (HEK293) cells by using perfusion with tangential flow filtration (TFF) and virus retention. For this purpose, six potential critical process parameters were evaluated using I-optimal design of experiments (DoE). The study showed that several input parameters and their interactions have significant impact on the infectious titer. Increasing the seeding cell density significantly improved the infectious titer, allowing infection at up to 46.6 × 10 6 cells mL -1 without decrease in the cell-specific virus yield. Keeping the perfusion pause after infection at minimum (1.1–1.3 h) and subsequently start the perfusion with a higher exchange rate (0.045–0.051 nL cell -1 d -1 ) was shown to be beneficial. The process was sensitive to shear stress and thus, the optimal crossflow rate was between 44 and 55 mL min -1 , which corresponds to 950–1150 s -1 shear rate. By optimizing the perfusion process, the titer reached up to 5.1 × 10 10 TCID 50 mL -1 , which is 17-fold higher than in batch cultivation. Overall, this work presents perfusion cultivation as an efficient technology to improve the VSV-GP titer with virus retention.

Compared to free antigens, antigens immobilized on scaffolds, such as nanoparticles, generally show improved immunogenicity. Conventionally, antigens are conjugated to scaffolds through genetic fusion or chemical conjugation, which may result in impaired assembly or heterogeneous binding and orientation of the antigens. By combining two emerging technologies—i.e., self-assembling multimeric protein scaffold particles (MPSPs) and bacterial superglue—these shortcomings can be overcome and antigens can be bound on particles in their native conformation. In the present work, we assessed whether this technology could improve the immunogenicity of a candidate subunit vaccine against the zoonotic Rift Valley fever virus (RVFV). For this, the head domain of glycoprotein Gn, a known target of neutralizing antibodies, was coupled on various MPSPs to further assess immunogenicity and efficacy in vivo. The results showed that the Gn head domain, when bound to the lumazine synthase-based MPSP, reduced mortality in a lethal mouse model and protected lambs, the most susceptible RVFV target animals, from viremia and clinical signs after immunization. Furthermore, the same subunit coupled to two other MPSPs (Geobacillus stearothermophilus E2 or a modified KDPG Aldolase) provided full protection in lambs as well.

Ongoing outbreaks of Middle East respiratory syndrome coronavirus (MERS-CoV) continue posing a global health threat. Vaccination of livestock reservoir species is a recommended strategy to prevent spread of MERS-CoV among animals and potential spillover to humans. Using a direct-contact llama challenge model that mimics naturally occurring viral transmission, we tested the efficacy of a multimeric receptor binding domain (RBD) particle-display based vaccine candidate. While MERS-CoV was transmitted to naïve animals exposed to virus-inoculated llamas, immunization induced robust virus-neutralizing antibody responses and prevented transmission in 1/3 vaccinated, in-contact animals. Our exploratory study supports further improvement of the RBD-based vaccine to prevent zoonotic spillover of MERS-CoV.

Ongoing outbreaks of Middle East respiratory syndrome coronavirus (MERS-CoV) continue posing a global health threat. Vaccination of livestock reservoir species is a recommended strategy to prevent spread of MERS-CoV among animals and potential spillover to humans. Using a direct-contact llama challenge model that mimics naturally occurring viral transmission, we tested the efficacy of a multimeric receptor binding domain (RBD) particle-display based vaccine candidate. While MERS-CoV was transmitted to naïve animals exposed to virus-inoculated llamas, immunization induced robust virus-neutralizing antibody responses and prevented transmission in 1/3 vaccinated, in-contact animals. Our exploratory study supports further improvement of the RBD-based vaccine to prevent zoonotic spillover of MERS-CoV.

Large-scale insect cell culture utilizing the baculovirus expression vector system (BEVS) can be used to produce biopharmaceuticals such as vaccines and therapeutic proteins. Biopharmaceutical production processes are generally complex and sensitive to many process parameters and changes, but on-line monitoring in this area is relatively limited and the fundamental understanding of the intricate relationships between significant process parameters and the process outcome, especially on the multi-liter or multi-m3 scale, is rarely conclusive. Dielectric spectroscopy (DS), which is based on the frequency dependent measurement of the passive dielectric properties of materials, was applied to large-scale insect cell cultures infected with a baculovirus under low multiplicity of infection conditions to produce a recombinant protein of the virus-like particle class. DS not only allowed the qualitative monitoring of the infection and recombinant protein production process within the culture in real-time but also the detection of important culture events, e.g. the peak in baculovirus production/concentration. Additionally, DS seemed to be able to serve as a predictive tool for the overall recombinant protein yield early in the process. Partial Least Square models were successfully developed allowing monitoring of the cultures progress in terms of cell density, size, and even nutrient concentration replacing the need for discrete sampling and therefore reducing contamination risks. In summary, DS has been demonstrated to have the potential to increase bioprocess understanding and the repeatability of recombinant protein production in the BEVS but ultimately also to satisfy the increased requirements for process monitoring as delineated recently in the Process Analytical Technology initiative by the Food and Drug Administration

Disturbed activation or regulation of the stress response through the hypothalamic-pituitary-adrenal (HPA) axis is a fundamental component of multiple stress-related diseases, including psychiatric, metabolic, and immune disorders. The FK506 binding protein 51 (FKBP5) is a negative regulator of the glucocorticoid receptor (GR), the main driver of HPA axis regulation, and FKBP5 polymorphisms have been repeatedly linked to stress-related disorders in humans. However, the specific role of Fkbp5 in the paraventricular nucleus of the hypothalamus (PVN) in shaping HPA axis (re)activity remains to be elucidated. We here demonstrate that the deletion of Fkbp5 in Sim1+ neurons dampens the acute stress response and increases GR sensitivity. In contrast, Fkbp5 overexpression in the PVN results in a chronic HPA axis over-activation, and a PVN-specific rescue of Fkbp5 expression in full Fkbp5 KO mice normalizes the HPA axis phenotype. Single-cell RNA sequencing revealed the cell-type-specific expression pattern of Fkbp5 in the PVN and showed that Fkbp5 expression is specifically upregulated in Crh+ neurons after stress. Finally, Crh-specific Fkbp5 overexpression alters Crh neuron activity, but only partially recapitulates the PVN-specific Fkbp5 overexpression phenotype. Together, the data establish the central and cell-type-specific importance of Fkbp5 in the PVN in shaping HPA axis regulation and the acute stress response.

Introduction: While vitamin deficiency after bariatric surgery has been repeatedly described, few studies have focused on adequate micronutrient status. In this study, we examine the prevalence of vitamin and micronutrient deficiency for the first 3 years after surgery. Methods: Out of 1,216 patients undergoing surgery, 485 who underwent postoperative follow-up in an outpatient clinic between 2010 and 2019 were included in this evaluation (76.9% women, mean age 42 ± 12 years, mean BMI: year 1, 33.9 ± 19.2; year 2, 29.7 ± 8.7; year 3, 26.2 ± 4.0). Weight and cardiovascular risk factors as well as ferritin, vitamin B12, folic acid, 25-OH-vitamin D, vitamin A, vitamin E, zinc, copper, and selenium were evaluated. Deficits were defined as follows: ferritin <15 µg/L, vitamin B12 <197 pg/mL, folic acid <4.4 ng/mL, 25-OH-vitamin D <75 nmol/L, vitamin A <1.05 µmol/L, vitamin E <12 µmol/L, zinc <0.54 mg/L, copper <0.81 mg/L, and selenium <50 µg/L. All patients underwent dietary counselling and substitution of the respective deficits as appropriate. Results: One year after bariatric surgery, 485 patients completed follow-up. This number decreased to 114 patients in year 2, and 80 patients in year 3. Overall, 42.7% (n = 207) underwent sleeve gastrectomy, 43.7% (n = 211) Roux-en-Y-gastric bypass, and 13.9% (n = 67) gastric banding. The following deficits were found (year 1/2/3): ferritin, 21.6/35.0/32.5%; vitamin B12, 14.3/1.8/6.3%; folic acid, 29.7/21.6/15.3%; 25-OH-vitamin D, 70.8/67.0/57.4%; vitamin A, 13.2/8.9/12.8%; vitamin E, 0%; zinc, 1.7/0/1.5%; copper, 10.4/12.2/11.9%; selenium, 11.1/4.3/0%. Conclusion: As seen in other studies, the follow-up frequency decreased over the years. Despite intensive substitution, the extent of some deficiencies increased or did not improve. These results suggest reinforcing measures to motivate patients for regular follow-up visits, considering closer monitoring schedules, and improving supplementation strategies.

Magnetic resonance spectroscopy (MRS) from voxels placed in the left anterior cingulate cortex (ACC) was measured from 14 boys with Autism Spectrum Disorder (ASD) and 24 gender and age-matched typically developing (TD) control group. Our main aims were to compare the concentration of γ-aminobutyric acid (GABA) between the two groups, and to investigate the relationship between brain metabolites and autism symptom severity in the ASD group. We did find a significant negative correlation in the ASD group between Autism Spectrum Screening Questionnaire (ASSQ) and GABA+/Cr, which may imply that severity of symptoms in ASD is associated with differences in the level of GABA in the brain, supporting the excitatory/inhibitory (E/I) imbalance theory. However we did not find a significant difference between the two groups in GABA levels.

Background Inherent resistance to radio/chemotherapy is one of the major reasons for early recurrence, treatment failure, and dismal prognosis of glioblastoma. Thus, the identification of resistance driving regulators as prognostic and/or predictive markers as well as potential vulnerabilities for combined modality treatment approaches is of pivotal importance. Methods We performed an integrative analysis of treatment resistance and DNA damage response regulator expression in a panel of human glioblastoma cell lines. mRNA expression levels of 38 DNA damage response regulators were analyzed by qRT-PCR. Inherent resistance to radiotherapy (single-shot and fractionated mode) and/or temozolomide treatment was assessed by clonogenic survival assays. Resistance scores were extracted by dimensionality reduction and subjected to correlation analyses with the mRNA expression data. Top-hit candidates with positive correlation coefficients were validated by pharmacological inhibition in clonogenic survival assays and DNA repair analyses via residual γH2AX/53BP1-foci staining. Results Inherent resistance to single-shot and similarly also to fractionated radiotherapy showed strong positive correlations with mRNA expression levels of known vulnerabilities of GBM, including PARP1, NBN, and BLM, as well as ATR and LIG4—two so far underestimated targets. Inhibition of ATR by AZD-6738 resulted in robust and dose-dependent radiosensitization of glioblastoma cells, whereas LIG4 inhibition by L189 had no noticeable impact. Resistance against temozolomide showed strong positive correlation with mRNA expression levels of MGMT as to be expected. Interestingly, it also correlated with mRNA expression levels of ATM, suggesting a potential role of ATM in the context of temozolomide resistance in glioblastoma cells. ATM inhibition exhibited slight sensitization effects towards temozolomide treatment in MGMT low expressing glioblastoma cells, thus encouraging further characterization. Conclusions Here, we describe a systematic approach integrating clonogenic survival data with mRNA expression data of DNA damage response regulators in human glioblastoma cell lines to identify markers of inherent therapy resistance and potential vulnerabilities for targeted sensitization. Our results provide proof-of-concept for the feasibility of this approach, including its limitations. We consider this strategy to be adaptable to other cancer entities as well as other molecular data qualities, and its upscaling potential in terms of model systems and observational data levels deserves further investigation.

Absence seizures (ASs) are characterized by pathological electrographic oscillations in the cerebral cortex and thalamus, which are called spike-and-wave discharges (SWDs). Subcortical structures, such as the cerebellum, may well contribute to the emergence of ASs, but the cellular and molecular underpinnings remain poorly understood. Here we show that the genetic ablation of P/Q-type calcium channels in cerebellar granule cells (quirky) or Purkinje cells (purky) leads to recurrent SWDs with the purky model showing the more severe phenotype. The quirky mouse model showed irregular action potential firing of their cerebellar nuclei (CN) neurons as well as rhythmic firing during the wave of their SWDs. The purky model also showed irregular CN firing, in addition to a reduced firing rate and rhythmicity during the spike of the SWDs. In both models, the incidence of SWDs could be decreased by increasing CN activity via activation of the G q -coupled designer receptor exclusively activated by designer drugs (DREADDs) or via that of the G q -coupled metabotropic glutamate receptor 1. In contrast, the incidence of SWDs was increased by decreasing CN activity via activation of the inhibitory G i/o -coupled DREADD. Finally, disrupting CN rhythmic firing with a closed-loop channelrhodopsin-2 stimulation protocol confirmed that ongoing SWDs can be ceased by activating CN neurons. Together, our data highlight that P/Q-type calcium channels in cerebellar granule cells and Purkinje cells can be relevant for epileptogenesis, that G q -coupled activation of CN neurons can exert anti-epileptic effects and that precisely timed activation of the CN can be used to stop ongoing SWDs.