Unraveling the Prefrontal Cortex-Basolateral Amygdala Pathway's Role on Schizophrenia's Cognitive Impairments: A Multimodal Study in Patients and Mouse Models.

BACKGROUND AND HYPOTHESIS: This study investigated the role of the medial prefrontal cortex (mPFC)-basolateral amygdala (BLA) pathway in schizophrenia (SCZ)-related cognitive impairments using various techniques. STUDY DESIGN: This study utilized clinical scales, magnetic resonance imaging, single-cell RNA sequencing, and optogenetics to investigate the mPFC-BLA pathway in SCZ patients. In the mouse model, 6-week-old methylazoxymethanol acetate-induced mice demonstrated significant cognitive deficits, which were addressed through stereotaxic injections of an adeno-associated viral vector to unveil the neural connection between the mPFC and BLA. STUDY RESULTS: Significant disparities in brain volume and neural activity, particularly in the dorsolateral prefrontal cortex (DLPFC) and BLA regions, were found between SCZ patients and healthy controls. Additionally, we observed correlations indicating that reduced volumes of the DLPFC and BLA were associated with lower cognitive function scores. Activation of the mPFC-BLA pathway notably improved cognitive performance in the SCZ model mice, with the targeting of excitatory or inhibitory neurons alone failing to replicate this effect. Single-cell transcriptomic profiling revealed gene expression differences in excitatory and inhibitory neurons in the BLA of SCZ model mice. Notably, genes differentially expressed in the BLA of these model mice were also found in the blood exosomes of SCZ patients. CONCLUSIONS: Our research provides a comprehensive understanding of the role of the PFC-BLA pathway in SCZ, underscoring its significance in cognitive impairment and offering novel diagnostic and therapeutic avenues. Additionally, our research highlights the potential of blood exosomal mRNAs as noninvasive biomarkers for SCZ diagnosis, underscoring the clinical feasibility and utility of this method.

Cytisine-N-methylene-(5,7,4'-trihydroxy)- isoflavone ameliorates ischemic stroke-induced brain injury in mouse by regulating the oxidative stress and BDNF-Trkb/Akt pathway.

BACKGROUND: A novel compound Cytisine-N-methylene-(5,7,4'-trihydroxy)- isoflavone (LY01) found in the Sophora alopecuroides L is a neuroprotective agent. However, the effect and potential mechanism of LY01 treatment for ischemic stroke (IS) have not been fully elucidated. AIM OF THE STUDY: The aim of this study is to demonstrate whether LY01 can rescue ischemic stroke-induced brain injury and oxygen-glucose deprivation/reperfusion (OGD/R). RESULTS: Our results show that intragastric administration of LY01 improves ischemic stroke behaviors in mice, as demonstrated by neurological score, infarct volume, cerebral water content, rotarod test for activity. Compared with the model group, the ginkgo biloba extract (EGb) and LY01 reversed the neurological score, infarct volume, cerebral water content, rotarod test in model mice. Further analysis showed that the LY01 rescued oxidative stress in the model mice, which was reflected in the increased levels of catalase, superoxide dismutase, total antioxidant capacity and decreased levels of malondialdehyde in the serum of the model mice. Moreover, the expression of the brain-derived neurotrophic factor brain-derived neurotrophic factor (BDNF), phosphorylated protein kinase B (p-Akt), Bax, Bcl-2, (p)-tropomysin related kinase B (p-Trkb) was restored and the expression of Bax, glial fibrillary acidic protein (GFAP) in the brains of the model mice was inhibited through LY01 treatment. In the polymerase chain reaction (PCR) data, after giving LY01, the expression in the brains of model mice was that, IL-10 increased and IL-1ß, Bax, Bcl-2 decreased. Furthermore, the results indicated that LY01 improved cell viability, reactive oxygen species content, and mitochondrial membrane potential dissipation induced by OGD/R in primary culture of rat cortical neurons. Bax and caspase-3 activity was upregulated compared to the before after treatment with LY01. CONCLUSIONS: Our study suggests that LY01 reversed ischemic stroke by reducing oxidative stress and activating the BDNF-TrkB/Akt pathway and exerted a neuroprotective action against OGD/R injury via attenuation, a novel approach was suggested to treat ischemic stroke. Our observations justify the traditional use of LY01 for a treatment of IS in nervous system.

A Novel Homozygous Mutation Causing Complete TYK2 Deficiency, with Severe Respiratory Viral Infections, EBV-Driven Lymphoma, and Jamestown Canyon Viral Encephalitis.

Autosomal recessive tyrosine kinase 2 (TYK2) deficiency is characterized by susceptibility to mycobacterial and viral infections. Here, we report a 4-year-old female with severe respiratory viral infections, EBV-driven Burkitt-like lymphoma, and infection with the neurotropic Jamestown Canyon virus. A novel, homozygous c.745C > T (p.R249*) variant was found in TYK2. The deleterious effects of the TYK2 lesion were confirmed by immunoblotting; by evaluating functional responses to IFN-α/ß, IL-10, and IL-23; and by assessing its scaffolding effect on the cell surface expression of cytokine receptor subunits. The effects of the mutation could not be pharmacologically circumvented in vitro, suggesting that alternative modalities, such as hematopoietic stem cell transplantation or gene therapy, may be needed. We characterize the first patient from Canada with a novel homozygous mutation in TYK2.

Ameliorative effects of escin on neuropathic pain induced by chronic constriction injury of sciatic nerve.

ETHNOPHARMACOLOGY RELEVANCE: Escin is a natural mixture of triterpene saponins extracted from the seeds of Aesculus wilsonii Rehd. And has been reported to possess the therapeutic effects against neuropathic pain (NP). However, the underlying mechanisms remain unclear. AIM OF THE STUDY: The present study aimed to investigate the therapeutic effects and explore the underlying mechanisms of escin on rats of NP induced by chronic constriction injury (CCI) of sciatic nerve. MATERIALS AND METHODS: Rats were treated with escin (7, 14, and 28 mg/kg, i. g.) daily from the third day after the surgery (day 0) for consecutive 14 days. Regular behavior and thermal threshold were measured on days 0, 3, 5, 7, 10 and 14. Investigations into mechanisms involved measurement of inflammatory factors and biochemical factors in dorsal root ganglion (DRG). Inflammatory pain responses and nerve injuries were induced by the CCI model. Tonic pain model and acute inflammatory model induced by formalin or carrageenan were established to evaluated the pharmacological effects of escin on acute inflammatory pain. Corresponding behaviors were monitored and relevant gene expression such as c-fos, mu opioid receptor (MOR) and KCNK1 were detected by qRT-PCR. Investigate the neuroprotective effects of escin on PC12 cell injury induced by lipopolysaccharide (LPS). Cell morphology was observed under inverted microscope and neuroprotective effect of escin on cell activity was assessed by MTT assay. RESULTS: Escin could widen thermal threshold, downregulate the concentration of inflammatory factors like tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, suppress the gene expression of toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), decrease the level of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) remarkably. In addition, escin significantly lowered the duration of licking, numbers of flinches and increase in paw edema, showing great therapeutic effects on inflammatory pain responses. Moreover, the activity of injured PC12 cells was significantly improved after escin administrated. CONCLUSION: Escin exerted the ameliorative effects on NP induced by CCI which may be related to downregulating the release of pro-inflammatory cytokines, suppressing TLR-4/NF-κB signal pathway, thereafter decreasing the level of GFAP and NGF.

Anti-invasion and anti-metastasis effects of Valjatrate E via reduction of matrix metalloproteinases expression and suppression of MAPK/ERK signaling pathway.

Valjatrate E is an iridoid compound extracted from Valeriana jatamansi Jones herb and is the active ingredient in antitumor activity. Here, we reported its action on tumor invasion and metastasis in the human hepatocellular carcinoma HepG2, aiming at a better understanding of the potential mechanism of action of Valjatrate E. HepG2 cells were treated with Valjatrate E at different concentrations. Wound healing assay and transwell chamber assay were used to determine the effects of Valjatrate E on the migration and invasiveness of HepG2 cells, respectively. Moreover, homogeneity and heterotypic adhesion experiments evaluated the adhesion property of HepG2 cells. The molecular mechanisms by which Valjatrate E inhibited the invasion and migration of HepG2 cells were investigated by gelatin zymography experiment and western blot. Treatment with Valjatrate E inhibited the migration and invasion of HepG2 cells. It achieved this by reducing the expression of matrix metalloprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9), by inhibition of heterogeneous adhesion ability, by blocking mitogen-activated protein kinase (MAPK) signaling via inhibiting the phosphorylation of extracellular signal-regulated kinases (p-ERK). Taken together, these findings provide new evidence that mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) signaling pathway plays an important role in promoting invasion and metastasis in HepG2 cells through p-ERK, and MAPK/ERK signaling pathway may be a therapeutic target for tumor.

Blockade of surface-bound TGF-ß on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment.

Regulatory T cells (Tregs) suppress antitumor immunity by inhibiting the killing of tumor cells by antigen-specific CD8+ T cells. To better understand the mechanisms involved, we used ex vivo three-dimensional collagen-fibrin gel cultures of dissociated B16 melanoma tumors. This system recapitulated the in vivo suppression of antimelanoma immunity, rendering the dissociated tumor cells resistant to killing by cocultured activated, antigen-specific T cells. Immunosuppression was not observed when tumors excised from Treg-depleted mice were cultured in this system. Experiments with neutralizing antibodies showed that blocking transforming growth factor-ß (TGF-ß) also prevented immunosuppression. Immunosuppression depended on cell-cell contact or cellular proximity because soluble factors from the collagen-fibrin gel cultures did not inhibit tumor cell killing by T cells. Moreover, intravital, two-photon microscopy showed that tumor-specific Pmel-1 effector T cells physically interacted with tumor-resident Tregs in mice. Tregs isolated from B16 tumors alone were sufficient to suppress CD8+ T cell-mediated killing, which depended on surface-bound TGF-ß on the Tregs Immunosuppression of CD8+ T cells correlated with a decrease in the abundance of the cytolytic protein granzyme B and an increase in the cell surface amount of the immune checkpoint receptor programmed cell death protein 1 (PD-1). These findings suggest that contact between Tregs and antitumor T cells in the tumor microenvironment inhibits antimelanoma immunity in a TGF-ß-dependent manner and highlight potential ways to inhibit intratumoral Tregs therapeutically.

Confocal microscopy with strip mosaicing for rapid imaging over large areas of excised tissue.

Confocal mosaicing microscopy is a developing technology platform for imaging tumor margins directly in freshly excised tissue, without the processing required for conventional pathology. Previously, mosaicing on 12-×-12 mm² of excised skin tissue from Mohs surgery and detection of basal cell carcinoma margins was demonstrated in 9 min. Last year, we reported the feasibility of a faster approach called "strip mosaicing," which was demonstrated on a 10-×-10 mm² of tissue in 3 min. Here we describe further advances in instrumentation, software, and speed. A mechanism was also developed to flatten tissue in order to enable consistent and repeatable acquisition of images over large areas. We demonstrate mosaicing on 10-×-10 mm² of skin tissue with 1-µm lateral resolution in 90 s. A 2.5-×-3.5 cm² piece of breast tissue was scanned with 0.8-µm lateral resolution in 13 min. Rapid mosaicing of confocal images on large areas of fresh tissue potentially offers a means to perform pathology at the bedside. Imaging of tumor margins with strip mosaicing confocal microscopy may serve as an adjunct to conventional (frozen or fixed) pathology for guiding surgery.

Detection of intra-tumor self antigen recognition during melanoma tumor progression in mice using advanced multimode confocal/two photon microscope.

Determining how tumor immunity is regulated requires understanding the extent to which the anti-tumor immune response "functions" in vivo without therapeutic intervention. To better understand this question, we developed advanced multimodal reflectance confocal/two photon fluorescence intra-vital imaging techniques to use in combination with traditional ex vivo analysis of tumor specific T cells. By transferring small numbers of melanoma-specific CD8+ T cells (Pmel-1), in an attempt to mimic physiologic conditions, we found that B16 tumor growth alone was sufficient to induce naive Pmel-1 T cell proliferation and acquisition of effector phenotype. Tumor -primed Pmel-1 T cells, are capable of killing target cells in the periphery and secrete IFNγ, but are unable to mediate tumor regression. Within the tumor, Pmel-1 T cells have highly confined mobility, displaying long term interactions with tumor cells. In contrast, adoptively transferred non tumor-specific OT-I T cells show neither confined mobility, nor long term interaction with B16 tumor cells, suggesting that intra-tumor recognition of cognate self antigen by Pmel-1 T cells occurs during tumor growth. Together, these data indicate that lack of anti-tumor efficacy is not solely due to ignorance of self antigen in the tumor microenvironment but rather to active immunosuppressive influences preventing a protective immune response.

Rapid confocal imaging of large areas of excised tissue with strip mosaicing.

Imaging large areas of tissue rapidly and with high resolution may enable rapid pathology at the bedside. The limited field of view of high-resolution microscopes requires the merging of multiple images that are taken sequentially to cover a large area. This merging or mosaicing of images requires long acquisition and processing times, and produces artifacts. To reduce both time and artifacts, we developed a mosaicing method on a confocal microscope that images morphology in large areas of excised tissue with sub-cellular detail. By acquiring image strips with aspect ratios of 10:1 and higher (instead of the standard ~1:1) and "stitching" them in software, our method images 10 × 10 mm(2) area of tissue in about 3 min. This method, which we call "strip mosaicing," is currently three times as fast as our previous method.

Area 3a neuron response to skin nociceptor afferent drive.

Area 3a neurons are identified that respond weakly or not at all to skin contact with a 25-38 degrees C probe, but vigorously to skin contact with the probe at > or =49 degrees C. Maximal rate of spike firing associated with 1- to 7-s contact at > or =49 degrees C occurs 1-2 s after probe removal from the skin. The activity evoked by 5-s contact with the probe at 51 degrees C remains above-background for approximately 20 s after probe retraction. After 1-s contact at 55-56 degrees C activity remains above-background for approximately 4 s. Magnitude of spike firing associated with 5-s contact increases linearly as probe temperature is increased from 49-51 degrees C. Intradermal capsaicin injection elicits a larger (approximately 2.5x) and longer-lasting (100x) increase in area 3a neuron firing rate than 5-s contact at 51 degrees C. Area 3a neurons exhibit enhanced or novel responsivity to 25-38 degrees C contact for a prolonged time after intradermal injection of capsaicin or alpha, beta methylene adenosine triphosphate. Their 1) delayed and persisting increase in spike firing in response to contact at > or =49 degrees C, 2) vigorous and prolonged response to intradermal capsaicin, and 3) enhanced and frequently novel response to 25-38 degrees C contact following intradermal algogen injection or noxious skin heating suggest that the area 3a neurons identified in this study contribute to second pain and mechanical hyperalgesia/allodynia.

Confocal mosaicing microscopy in Mohs skin excisions: feasibility of rapid surgical pathology.

Mosaicing of confocal images enables observation of nuclear morphology in large areas of tissue. An application of interest is rapid detection of basal cell carcinomas (BCCs) in skin excisions during Mohs surgery. A mosaic is currently created in less than 9 min, whereas preparing frozen histology requires 20 to 45 min for an excision. In reflectance mosaics, using acetic acid as a contrast agent to brighten nuclei, large and densely nucleated BCC tumors were detectable in fields of view of 12 x 12 mm (which is equivalent to a 2x-magnified view as required by Mohs surgeons). However, small and sparsely nucleated tumors remained undetectable. Their diminutive size within the large field of view resulted in weak light backscatter and contrast relative to the bright surrounding normal dermis. In fluorescence, a nuclear-specific contrast agent may be used and light emission collected specifically from nuclei but almost none from the dermis. Acridine orange of concentration 1 mM stains nuclei in 20 s with high specificity and strongly enhances nuclear-to-dermis contrast of BCCs. Comparison of fluorescence mosaics to histology shows that both large and small tumors are detectable. The results demonstrate the feasibility of confocal mosaicing microscopy toward rapid surgical pathology to potentially expedite and guide surgery.

Confocal reflectance mosaicing of basal cell carcinomas in Mohs surgical skin excisions.

Precise removal of basal cell carcinomas (BCCs) with minimal damage to the surrounding normal skin is guided by the examination of frozen histology of each excision during Mohs surgery. The preparation of frozen histology is slow, requiring 20 to 45 min per excision. Confocal reflectance mosaicing may enable rapid detection of BCCs directly in surgical excisions, with minimal need for frozen histology. Soaking the excisions in acetic acid rapidly brightens nuclei and enhances BCC-to-dermis contrast. Clinically useful concentrations of acetic acid from 10 to 1% require 30 s to 5 min, respectively. A tissue fixture precisely controls the stability, flatness, tilt, and sag of the excisions, which enables mosaicing of 36x36 images to create a field of view of 12x12 mm. This simulates a 2x magnification view in light microscopes, which is routinely used by Mohs surgeons to examine frozen histology. Compared to brightfield, cross-polarization enhances contrast and detectability of BCCs in the papillary dermis but not in the reticular dermis. Comparison of mosaics to histology shows that nodular, micronodular, and superficial BCCs are easily detected. However, infiltrative and sclerosing BCCs tend to be obscured within the surrounding bright dermis. The mosaicing method currently requires 9 min, and thus may expedite Mohs surgery.

Dual mode reflectance and fluorescence confocal laser scanning microscopy for in vivo imaging melanoma progression in murine skin.

A system was designed and developed for simultaneous fluorescence and reflectance contrast in vivo confocal imaging of murine skin using 488 nm (fluorescence mode) and 830 nm (reflectance mode) laser light sources. B16 melanoma cells and B16-enhanced green fluorescent protein (EGFP) cells were inoculated intradermally into transgenic C57BL/6-TgN (ACTbEGFP) 10sb and non-transgenic C57BL/6 mice, respectively. The inoculation sites were imaged sequentially over a 20 d period. The in vivo confocal images were correlated with ex vivo conventional microscopy. The combined modality system provided single-cell resolution and adequate image registration. In fluorescence mode, B16 melanoma cells appeared as dark objects in the bright background of the GFP expressing murine cells of the C57BL/6 transgenic mouse, and the B16-EGFP melanoma cells had a bright signal within a dark background in C57BL/6 mice. In the C57BL/6 transgenic mouse, a population of fluorescent dendritic cells was observed in the vicinity of the tumor cells. The reflectance images provide a useful reference for those areas in the dermal tissues lacking a fluorescent signal. Combined reflectance/fluorescence in vivo confocal laser scanning microscopy holds significant promise for studies of tumor progression in murine skin.

Correcting common errors in identifying cancer-specific serum peptide signatures.

"Molecular signatures" are the qualitative and quantitative patterns of groups of biomolecules (e.g., mRNA, proteins, peptides, or metabolites) in a cell, tissue, biological fluid, or an entire organism. To apply this concept to biomarker discovery, the measurements should ideally be noninvasive and performed in a single read-out. We have therefore developed a peptidomics platform that couples magnetics-based, automated solid-phase extraction of small peptides with a high-resolution MALDI-TOF mass spectrometric readout (Villanueva, J.; Philip, J.; Entenberg, D.; Chaparro, C. A.; Tanwar, M. K.; Holland, E. C.; Tempst, P. Anal. Chem. 2004, 76, 1560-1570). Since hundreds of peptides can be detected in microliter volumes of serum, it allows to search for disease signatures, for instance in the presence of cancer. We have now evaluated, optimized, and standardized a number of clinical and analytical chemistry variables that are major sources of bias; ranging from blood collection and clotting, to serum storage and handling, automated peptide extraction, crystallization, spectral acquisition, and signal processing. In addition, proper alignment of spectra and user-friendly visualization tools are essential for meaningful, certifiable data mining. We introduce a minimal entropy algorithm, "Entropycal", that simplifies alignment and subsequent statistical analysis and increases the percentage of the highly distinguishing spectral information being retained after feature selection of the datasets. Using the improved analytical platform and tools, and a commercial statistics program, we found that sera from thyroid cancer patients can be distinguished from healthy controls based on an array of 98 discriminant peptides. With adequate technological and computational methods in place, and using rigorously standardized conditions, potential sources of patient related bias (e.g., gender, age, genetics, environmental, dietary, and other factors) may now be addressed.

Treatment of intracranial rat glioma model with implant of radiosensitizer and biomodulator drug combined with external beam radiotherapy.

PURPOSE: To evaluate an intracranial polymer implant containing bromodeoxyuridine (BrdUrd) and N-(phosphonacetyl)-L-aspartic acid (PALA) in combination with external beam radiotherapy (EBRT) in the treatment of a rat glioma. METHODS AND MATERIALS: Combinations of the biomodulators 5-fluorouracil, methotrexate, or PALA with BrdUrd were evaluated as radiosensitizers in vitro by clonogenic assay. In in vivo experiments, BrdUrd and PALA were incorporated into a polyanhydride-based polymer, bis(p-carboxyphenoxy)propane sebacic acid, and implanted in the C6 rat glioma growing intracranially. The effectiveness of treatment was evaluated on the basis of survival. EBRT was given as 10-MV X-rays. RESULTS: In tissue culture experiments, C6 cells were refractory to radiosensitization by BrdUrd even when the thymidine analog was combined with a biomodulator intended to reduce de novo thymidine synthesis. The most effective compound in vitro was PALA. When PALA and BrdUrd in a polymer formulation were implanted intracranially and combined with 10-Gy EBRT, the treatment was highly effective, with 83% of treated rats surviving 180 days. CONCLUSION: Although the in vitro results were not encouraging, the combination of intratumoral BrdUrd and PAL with 10-Gy EBRT was highly effective in treating a rat glioma. These results indicate the clinical potential of combined and mixed modality treatments involving intratumoral sustained-release drug delivery.