Non-overlapping cell populations within dorsomedial prefrontal cortex (dmPFC), defined by gene expression or projection target, control dissociable aspects of reward seeking through unique activity ...patterns. However, even within these defined cell populations, considerable cell-to-cell variability is found, suggesting that greater resolution is needed to understand information processing in dmPFC. Here, we use two-photon calcium imaging in awake, behaving mice to monitor the activity of dmPFC excitatory neurons throughout Pavlovian reward conditioning. We characterize five unique neuronal ensembles that each encodes specialized information related to a sucrose reward, reward-predictive cues, and behavioral responses to those cues. The ensembles differentially emerge across daily training sessions - and stabilize after learning - in a manner that improves the predictive validity of dmPFC activity dynamics for deciphering variables related to behavioral conditioning. Our results characterize the complex dmPFC neuronal ensemble dynamics that stably predict reward availability and initiation of conditioned reward seeking following cue-reward learning.
Multiphoton microscopy is one of several new technologies providing unprecedented insight into the activity dynamics and function of neural circuits. Unfortunately, some of these technologies require ...experimentation in head-restrained animals, limiting the behavioral repertoire that can be integrated and studied. This issue is especially evident in drug addiction research, as no laboratories have coupled multiphoton microscopy with simultaneous intravenous drug self-administration, a behavioral paradigm that has predictive validity for treatment outcomes and abuse liability. Here, we describe a new experimental assay wherein head-restrained mice will press an active lever, but not inactive lever, for intravenous delivery of heroin or cocaine. Similar to freely moving animals, we find that lever pressing is suppressed through daily extinction training and subsequently reinstated through the presentation of relapse-provoking triggers (drug-associative cues, the drug itself, and stressors). Finally, we show that head-restrained mice will show similar patterns of behavior for oral delivery of a sucrose reward, a common control used for drug self-administration experiments. Overall, these data demonstrate the feasibility of combining drug self-administration experiments with technologies that require head-restraint, such as multiphoton imaging. The assay described could be replicated by interested labs with readily available materials to aid in identifying the neural underpinnings of substance use disorder.
Aim
. To evaluate gender-related differences of left atrial (LA) phasic function and structural remodeling in conjunction with the parameters of left ventricular (LV) diastolic dysfunction and ...central aortic pressure in patients with hypertension and recurrent atrial fibrillation (AF).
Material and methods
. The comparative study included 30 men and 37 women with non-valvular AF, hypertension and LV hypertrophy. Conventional echocardiographic measures were extended with LA measures, including its volume in three phases, LAemptying fraction (LAEF), passive and active ejection fraction. The parameters of central aortic pressure were estimated by applanation tonometry method.
Results
. No difference was observed between LA and LV structural parameters in men and women. However, in women LAEF (39 28;50 vs 50 42;55%; p=0.02) and E/E’(9.7 7.8;12 vs 7.1 5.6;8.6; p=0.001) were worse than in men. Active LA ejection fraction was higher in women (31 21;42 vs 24 19;31%; p=0.04), whereas passive one – in men (12 10;14 vs 33 23;38%; p<0.001), respectively. Men and women had comparable heart rate (HR), central and peripheral systolic and diastolic pressure, pulse wave velocity (PWV), but women had higher augmentation index (AI) values 33 28;39 vs 23 21;28%; p<0.001, even adjusted by HR (AI 75) (34 27;39 vs 26 20;29%; p<0.001). Only in men PWV weakly correlated with AI 75 (r=0.44; p=0.02 versus r=-0.11; p=0.51, respectively for men and women; intergroup differences: z=2.26; p=0.012). In a multivariate regression analysis in men LAEF was significantly associated with height, weight, E’, E/E’ and glomerular filtration rate (GFR), whereas in women – with E’ and AI 75.
Conclusion
. Patients of different genders with recurrent AF and hypertension have comparable LA structuralremodeling. However, women characterized by a more pronounced decrease in LAEF and impaired LV diastolic function than men. In women as distinct from men LV filling is predominantly due to LA systole. In a multivariate regression analysis in men LAEF was significantly associated with height, weight, E’, E/E’ and GFR, whereas in women – with E’ and AI 75.
During the ongoing coronavirus disease COVID-19 pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An ...important unanswered question is what levels of T cell and antibody responses are sufficient to protect from the infection.
In 5340 Moscow residents, we evaluated anti-SARS-CoV-2 IgM/IgG titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using IFNγ ELISpot assay. Additionally, we evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFNγ and IL2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T cell responses, using the Kaplan-Meyer estimator method, for up to 300 days post-inclusion.
We showed that T cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, while the T cell response by itself granted only intermediate protection.
We found that the contribution of the virus-specific antibodies to protection against the SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized health care and public anti-COVID-19 policies.
EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5′...CC*T/AGG...3′ and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner ...cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2′-deoxyuridine (i
5
dU) or 5-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl-2′-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRH recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide- peptides obtained. DNA duplexes containing i
5
dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly
268
-Met
391
region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.
The purpose of this prospective pilot study was to evaluate the feasibility and effects of cognitive-motor intervention on the cognitive and motor abilities of pediatric survivors of posterior fossa ...tumors. The study involved patients aged 7 to 18 years with cognitive deficits who had completed primary treatment for posterior fossa tumors. 25 participants (M
age
=11.3 ± 2.93, 64% male; 17 medulloblastoma, 1 ependymoma, 1 desmoplastic medulloblastoma, 6 piloid astrocytoma; 22 in remission (M
months
=45), 3 in stabilization (M
months
=49)) were recruited from the Research Institute for Brain Development and Peak Performance. The intervention consisted of two phases with a 3-month break for home training, and a total duration of 6 months. Each phase lasted 7 weeks and included two assessment procedures (pre- and post-intervention) and 10 training sessions over a period of 5 weeks (two 3-hour sessions per week). At baseline and pre- and post-intervention, all participants underwent a battery of cognitive and motor tests. Each training session included gross motor training (GMT), graphomotor training (GT), and cognitive-motor training (CMT). Statistical analysis was performed using the Friedman test for repeated measures and post-hoc Durbin-Conover test. The results indicated significant improvements in visuospatial working memory, visual attention, eye-hand coordination, semantic verbal fluency, auditory-motor synchronization, reaction time, and a decrease in the rate of ataxia. These improvements remained stable even in the absence of direct intervention. The findings demonstrate positive effects and feasibility of the intervention and suggest the need for further research in this area including randomized controlled feasibility studies with a larger sample.
Objectives
The conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of ...tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non‐woven material in order to obtain a well‐differentiated human airway epithelium.
Materials and methods
The bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy.
Results
With the use of a copolymer of chitosan‐gelatin‐poly‐l‐lactide, a bilayered non‐woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed.
Conclusions
The structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well‐differentiated equivalent of respiratory epithelium on non‐woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co‐culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.
RNA interference (RNAi)-based therapeutics hold the potential for dominant genetic disorders, enabling sequence-specific inhibition of pathogenic gene products. We aimed to direct RNAi for the ...selective suppression of the heterozygous
c.607 G > A variant causing
encephalopathy. By screening short interfering RNA (siRNA), we showed that
c.607G>A is a druggable target for RNAi. The si1488 candidate achieved at least twofold allelic discrimination and downregulated mutant protein to 35%. We created vectorized RNAi by incorporating the si1488 sequence into the short hairpin RNA (shRNA) in the adeno-associated virus (AAV) vector. The shRNA stem and loop were modified to improve the transcription, processing, and guide strand selection. All tested shRNA constructs demonstrated selectivity toward mutant
, while tweaking hairpin structure only marginally affected the silencing efficiency. The selectivity of shRNA-mediated silencing was confirmed in the context of AAV vector transduction. To conclude, RNAi effectors ranging from siRNA to AAV-RNAi achieve suppression of the pathogenic
c.607G>A and discriminate alleles by the single-nucleotide substitution. For gene therapy development, it is crucial to demonstrate the benefit of these RNAi effectors in patient-specific neurons and animal models of the GNAO1 encephalopathy.
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