Semaphorin7A patterns neural circuitry in the lateral line of the zebrafish.

In a developing nervous system, axonal arbors often undergo complex rearrangements before neural circuits attain their final innervation topology. In the lateral line sensory system of the zebrafish, developing sensory axons reorganize their terminal arborization patterns to establish precise neural microcircuits around the mechanosensory hair cells. However, a quantitative understanding of the changes in the sensory arbor morphology and the regulators behind the microcircuit assembly remain enigmatic. Here, we report that Semaphorin7A (Sema7A) acts as an important mediator of these processes. Utilizing a semi-automated three-dimensional neurite tracing methodology and computational techniques, we have identified and quantitatively analyzed distinct topological features that shape the network in wild-type and Sema7A loss-of-function mutants. In contrast to those of wild-type animals, the sensory axons in Sema7A mutants display aberrant arborizations with disorganized network topology and diminished contacts to hair cells. Moreover, ectopic expression of a secreted form of Sema7A by non-hair cells induces chemotropic guidance of sensory axons. Our findings propose that Sema7A likely functions both as a juxtracrine and as a secreted cue to pattern neural circuitry during sensory organ development.

[Using Artificial Intelligence Software for Diagnosing Emphysema and Interstitial Lung Disease]. / íê¸°ì¢ ë° 간질성 폐질환: 인공지능 소프트웨어 사용 경험.

Researchers have developed various algorithms utilizing artificial intelligence (AI) to automatically and objectively diagnose patterns and extent of pulmonary emphysema or interstitial lung diseases on chest CT scans. Studies show that AI-based quantification of emphysema on chest CT scans reveals a connection between an increase in the relative percentage of emphysema and a decline in lung function. Notably, quantifying centrilobular emphysema has proven helpful in predicting clinical symptoms or mortality rates of chronic obstructive pulmonary disease. In the context of interstitial lung diseases, AI can classify the usual interstitial pneumonia pattern on CT scans into categories like normal, ground-glass opacity, reticular opacity, honeycombing, emphysema, and consolidation. This classification accuracy is comparable to chest radiologists (70%-80%). However, the results generated by AI are influenced by factors such as scan parameters, reconstruction algorithms, radiation doses, and the training data used to develop the AI. These limitations currently restrict the widespread adoption of AI for quantifying pulmonary emphysema and interstitial lung diseases in daily clinical practice. This paper will showcase the authors' experience using AI for diagnosing and quantifying emphysema and interstitial lung diseases through case studies. We will primarily focus on the advantages and limitations of AI for these two diseases.

Predicting motor gains with home-based telerehabilitation after stroke.

INTRODUCTION: Telerehabilitation (TR) may be useful for rehabilitation therapy after stroke. However, stroke is a heterogeneous condition, and not all patients can be expected to derive the same benefit from TR, underscoring the need to identify predictors of response to TR. METHODS: A prior trial provided patients with 6 weeks of intensive rehabilitation therapy targeting arm movement, randomly assigned to be provided in the home via TR (current focus) or in clinic. Eligible patients had moderate arm motor deficits and were in the subacute-chronic stage post stroke. Behavioral gains were measured as change in the arm motor Fugl-Meyer score from baseline to 30 days post therapy. To delineate predictors of TR response, multivariable linear regression was performed, advancing the most significant predictor from each of eight categories: patient demographics, stroke characteristics, medical history, rehabilitation therapy outside of study procedures, motivation, sensorimotor impairment, cognitive/affective deficits, and functional status. RESULTS: The primary focus was on patients starting TR >90 days post stroke onset (n = 44), among whom female sex, less spasticity, and less visual field defects predicted greater motor gains. This model explained 39.3% of the variance in treatment-related gains. In secondary analysis that also included TR patients enrolled ≤90 days post stroke (total n = 59), only female sex was a predictor of treatment gains. A separate secondary analysis examined patients >90 days post stroke (n = 34) randomized to in-clinic therapy, among whom starting therapy earlier post stroke and less ataxia predicted greater motor gains. DISCUSSION: Response to TR varies across patients, emphasizing the need to identify characteristics that predict treatment-related behavioral gain. The current study highlights factors that might be important to patient selection for home-based TR after stroke.

Mitochondrial dysfunction induced by callyspongiolide promotes autophagy-dependent cell death.

Callyspongiolide is a marine macrolide known to induce caspaseindependent cancer cell death. While its toxic effects have been known, the mechanism leading to cell death is yet to be identified. We report that Callyspongiolide R form at C-21 (cally2R) causes mitochondrial dysfunction by inhibiting mitochondrial complex I or II, leading to a disruption of mitochondrial membrane potential and a deprivation of cellular energy. Subsequently, we observed, using electron microscopy, a drastic formation of autophagosome and mitophagy. Supporting these data, LC3, an autophagosome marker, was shown to co-localize with LAMP2, a lysosomal protein, showing autolysosome formation. RNA sequencing results indicated the induction of hypoxia and blocking of EGF-dependent pathways, which could be caused by induction of autophagy. Furthermore, mTOR and AKT pathways preventing autophagy were repressed while AMPK was upregulated, supporting autophagosome progress. Finally, the combination of cally2R with known anti-cancer drugs, such as gefitinib, sorafenib, and rapamycin, led to synergistic cell death, implicating potential therapeutic applications of callyspongiolide for future treatments. [BMB Reports 2021; 54(4): 227-232].

Development of γ-aminobutyric acid-, glycine-, and glutamate-immunopositive boutons on the rat genioglossal motoneurons.

Detailed information about the development of excitatory and inhibitory synapses on the genioglossal (GG) motoneuron may help to understand the mechanism of fine control of GG motoneuron firing and the coordinated tongue movement during postnatal development. For this, we investigated the development of γ-aminobutyric acid (GABA)-immunopositive (GABA +), glycine + (Gly +), and glutamate + (Glut +) axon terminals (boutons) on the somata of rat GG motoneurons at a postnatal day 2 (P2), P6 and P18 by retrograde labeling of GG motoneurons with horseradish peroxidase, electron microscopic postembedding immunogold staining with GABA, Gly, and Glut antisera, and quantitative analysis. The number of boutons per GG motoneuron somata and the mean length of bouton apposition, measures of bouton size and synaptic covering percentage, were significantly increased from P2/P6 to P18. The number and fraction of GABA + only boutons of all boutons decreased significantly, whereas those of Gly + only boutons increased significantly from P2/P6 to P18, suggesting developmental switch from GABAergic to glycinergic synaptic transmission. The fraction of mixed GABA +/Gly + boutons of all boutons was the highest among inhibitory bouton types throughout the postnatal development. The fractions of excitatory and inhibitory boutons of all boutons remained unchanged during postnatal development. These findings reveal a distinct developmental pattern of inhibitory synapses on the GG motoneurons different from that on spinal or trigeminal motoneurons, which may have an important role in the regulation of the precise and coordinated movements of the tongue during the maturation of the oral motor system.

Glutamatergic synaptic input to glioma cells drives brain tumour progression.

A network of communicating tumour cells that is connected by tumour microtubes mediates the progression of incurable gliomas. Moreover, neuronal activity can foster malignant behaviour of glioma cells by non-synaptic paracrine and autocrine mechanisms. Here we report a direct communication channel between neurons and glioma cells in different disease models and human tumours: functional bona fide chemical synapses between presynaptic neurons and postsynaptic glioma cells. These neurogliomal synapses show a typical synaptic ultrastructure, are located on tumour microtubes, and produce postsynaptic currents that are mediated by glutamate receptors of the AMPA subtype. Neuronal activity including epileptic conditions generates synchronised calcium transients in tumour-microtube-connected glioma networks. Glioma-cell-specific genetic perturbation of AMPA receptors reduces calcium-related invasiveness of tumour-microtube-positive tumour cells and glioma growth. Invasion and growth are also reduced by anaesthesia and the AMPA receptor antagonist perampanel, respectively. These findings reveal a biologically relevant direct synaptic communication between neurons and glioma cells with potential clinical implications.

Anti­photoaging and anti­oxidative activities of natural killer cell conditioned medium following UV­B irradiation of human dermal fibroblasts and a reconstructed skin model.

Conditioned media from various sources comprise numerous growth factors and cytokines and are known to promote the regeneration of damaged tissues. Among these, natural killer cell conditioned medium (NK­CdM) has been shown to stimulate collagen synthesis and the migration of fibroblasts during the wound healing process. With a long­term aim of developing a treatment for skin photoaging, the ability of NK­CdM to prevent ultraviolet­B (UV­B) damage was assessed in neonatal human dermal fibroblasts (NHDFs) and an in vitro reconstructed skin model. The factors present in NK­CdM were profiled using an antibody array analysis. Protein and mRNA levels in UV­B exposed NHDFs treated with NK­CdM were measured by western blotting and quantitative reverse transcription­PCR, respectively. The total antioxidant capacity of NK­CdM was determined to assess its ability to suppress reactive oxygen species. The anti­photoaging effect of NK­CdM was also assessed in a 3D reconstituted human full skin model. NK­CdM induced proliferation of UV­B­treated NHDFs, increased procollagen expression, and decreased matrix metalloproteinase (MMP)­1 expression. NK­CdM also exhibited a potent antioxidant activity as measured by the total antioxidant capacity. NK­CdM inhibited UV­B­induced collagen degradation by inactivating MAPK signaling. NK­CdM also elicited potential anti­wrinkle effects by inhibiting the UV­B­induced increase in MMP­1 expression levels in a 3D reconstituted human full skin model. Taken together, the suppression of both UV­B­induced MMP­1 expression and JNK activation by NK­CdM suggests NK­CdM as a possible candidate anti­skin aging agent.

Distribution of excitatory and inhibitory axon terminals on the rat hypoglossal motoneurons.

Detailed information about the excitatory and inhibitory synapses on the hypoglossal motoneurons may help understand the neural mechanism for control of the hypoglossal motoneuron excitability and hence the precise and coordinated movements of the tongue during chewing, swallowing and licking. For this, we investigated the distribution of GABA-, glycine (Gly)- and glutamate (Glut)-immunopositive (+) axon terminals on the genioglossal (GG) motoneurons by retrograde tracing, electron microscopic immunohistochemistry, and quantitative analysis. Small GG motoneurons (< 400 µm2 in cross-sectional area) had fewer primary dendrites, significantly higher nuclear/cytoplasmic ratio, and smaller membrane area covered by synaptic boutons than large GG motoneurons (> 400 µm2). The fraction of inhibitory boutons (GABA + only, Gly + only, and mixed GABA +/Gly + boutons) of all boutons was significantly higher for small GG motoneurons than for large ones, whereas the fraction of Glut + boutons was significantly higher for large GG motoneurons than for small ones. Almost all boutons (> 95%) on both small and large GG motoneurons were GABA + , Gly + or Glut + . The frequency of mixed GABA +/Gly + boutons was the highest among inhibitory boutons types for both small and large GG motoneurons. These findings may elucidate the anatomical substrate for precise regulation of the motoneuron firing required for the fine movements of the tongue, and also suggest that the excitability of small and large GG motoneurons may be regulated differently.

Quality by Design characterization of the perfusion culture process for recombinant FVIII.

A Quality by Design (QbD) concept was applied to characterize a cell culture process for production of the recombinant Factor VIII (rFVIII). We characterized the production bioreactor process and defined the design space by applying risk assessment to determine potential critical process parameters (CPPs) impacting critical quality attributes (CQAs). Characterization studies were subsequently performed using a qualified scaled-down model (SDM) and a multi-factorial design of experiment (DOE) approach to determine both the individual and combined impacts of the potential CPPs on CQAs. Among the operating parameters characterized, production temperature, production pH and a shift in the timing of production affected rFVIII activity and tyrosine sulfation level. Finally, we identified CPPs and established a design space for the cell culture process to identify appropriate conditions for routine manufacturing.

Overproduction of recombinant E. coli malate synthase enhances Chlamydomonas reinhardtii biomass by upregulating heterotrophic metabolism.

High uptake of malate and efficient distribution of intracellular malate to organelles contributed to biomass increase, reducing maintenance energy. In this study, transgenic Chlamydomonas reinhardtii was developed that stably expresses malate synthase in the chloroplast. The strains under glyoxylate treatment showed 19% more increase in microalgal biomass than wild-type. By RNA analysis, transcript levels of malate dehydrogenase (MDH4) and acetyl-CoA synthetase (ACS3), isocitrate lyase (ICL1) and malate synthase (MAS1), were significantly more expressed (17%, 42%, 24%, and 18% respectively), which was consistent with reported heterotrophic metabolism flux analysis with the objective function maximizing biomass. Photosynthetic Fv/Fm was slightly reduced. A more meticulous analysis is necessary, but, in the transgenic microalgae with malate synthase overexpression, the metabolism is likely to more rely on heterotrophic energy production via TCA cycle and glyoxylate shunt than on photosynthesis, resulting in the increase in microalgal biomass.