Histochemical Staining Research Articles

The Contribution of Mast Cells to the Regulation of Elastic Fiber Tensometry in the Skin Dermis of Children with Marfan Syndrome.

Marfan syndrome (MFS) is a hereditary condition accompanied by disorders in the structural and regulatory properties of connective tissue, including elastic fibers, due to a mutation in the gene encodes for fibrillin-1 protein (FBN1 gene) and the synthesis of abnormal fibrillin-1 glycoprotein. Despite the high potential of mast cells (MCs) to remodel the extracellular matrix (ECM), their pathogenetic significance in MFS has not been considered yet. The group of patients with Marfan syndrome included two mothers and five children (three girls aged 4, 11, and 11 and two boys aged 12 and 13). Normal skin was examined in two children aged 11 and 12. Histochemical, monoplex, and multiplex immunohistochemical techniques; combined protocols of simultaneous histochemical and immunohistochemical staining (the results of staining were assessed using light, epifluorescence, and confocal microscopy); and bioinformatics algorithms for the quantitative analysis of detected targets were used to evaluate mast cells and their relationship with other cells from extracellular structures in the skin dermis. Analysis of the skin MC population in children with Marfan syndrome revealed a considerably increased number of intra-organic populations with the preservation of the specific Tryptase+Chymase+CPA3+ protease profile typical of the skin. The features of the MC histotopography phenotype in MFS consisted of closer colocalization with elastic fibers, smooth muscle cells, and fibroblasts. MCs formed many intradermal clusters that synchronized the activity of cell functions in the stromal landscape of the tissue microenvironment with the help of spatial architectonics, including the formation of cell chains and the creation of fibrous niches. In MCs, the expression of specific proteases, TGF-β, and heparin increased, with targeted secretion of biologically active substances relative to the dermal elastic fibers, which had specific structural features in MFS, including abnormal variability in thickness along their entire length, alternating thickened and thinned areas, and uneven surface topography. This paper discusses the potential role of MCs in strain analysis (tensometry) of the tissue microenvironment in MFS. Thus, the quantitative and qualitative rearrangements of the skin MC population in MFS are aimed at altering the stromal landscape of the connective tissue. The results obtained should be taken into account when managing clinical signs of MFS manifested in other pathogenetically critical structures of internal organs, including the aorta, tendons, cartilage, and parenchymal organs.

A Histological Analysis and Detection of Complement Regulatory Protein CD55 in SARS-CoV-2 Infected Lungs.

A complement imbalance in lung alveolar tissue can play a deteriorating role in COVID-19, leading to acute respiratory distress syndrome (ARDS). CD55 is a transmembrane glycoprotein that inhibits the activation of the complement system at the intermediate cascade level, blocking the activity of the C3 convertase. In our study, lung specimens from COVID-19 and ARDS-positive COVID+/ARDS+ patients were compared with COVID-19 and ARDS-negative COVID-/ARDS- as well as COVID-/ARDS+ patients. Histochemical staining and immunolabeling of CD55 protein were performed. The COVID-/ARDS- specimen showed higher expression and homogeneous distribution of glycosaminoglycans as well as compactly arranged elastic and collagen fibers of the alveolar walls in comparison to ARDS-affected lungs. In addition, COVID-/ARDS- lung tissues revealed stronger and homogenously distributed CD55 expression on the alveolar walls in comparison to the disrupted COVID-/ARDS+ lung tissues. Even though the collapse of the alveolar linings and the accumulation of cellular components in the alveolar spaces were characteristic of COVID+/ARDS+ lung tissues, evaluating CD55 expression could be relevant to understand its relation to the disease. Furthermore, targeting CD55 upregulation as a potential therapy could be an option for post-infectious complications of COVID-19 and other inflammatory lung diseases in the future.

Aspartic proteases gene family: Identification and expression profiles during stem vascular development in tobacco

Aspartic proteases (APs) constitute a large family in plants and are widely involved in diverse biological processes, like chloroplast metabolism, biotic and abiotic stress responses, and reproductive development. In this study, we focused on overall analysis of the APs genes in tobacco. Our analysis included the phylogeny and cis-elements in the cell wall-associated promoters of these genes. To characterize the expression patterns of APs genes in stem vascular development. The tissue expression analysis showed that NtAED3-like was preferentially expressed in the differentiating xylem and phloem cells of the vascular system. Based on histochemical staining analysis showed that the NtAED3-like gene was specifically expressed in stem vascular tissue, root vascular tissue, and petiole vascular tissue. The TdT-mediated dUTP nick-end labeling (TUNEL) assay illustrated a delayed progression of programmed cell death (PCD) within the xylem of the ko-ntaed3a-like mutant, relative to the wild type. The mutant ko-ntaed3a-like exhibited a phenotype of thinning stem circumference and changed in xylem structure and lignin content. In addition, the two-dimension heteronuclear single quantum coherent nuclear magnetic resonance (2D-HSQC) analysis of three milled wood lignins (MWLs) showed that the content of β-O-4 connection in ko-ntaed3a-like decreased slightly compared with wild type. In conclusion, this study provides our understanding of the regulation of vascular tissue development by the NtAED3-like gene in tobacco and provides a better basis for determining the molecular mechanism of the aspartic protease in secondary cell wall (SCW) development.

Genome-wide analysis of the HD-Zip IV gene family in Camellia sinensis and functional characterization of CsHDZIV3 under trichome development

The HD-Zip IV transcription factors, which belong to the homeodomain leucine zipper (HD-ZIP) family, are essential for the development of epidermal cells in plants. However, there is limited information about HD-Zip IV in tea plant (Camellia sinensis L.). Here we identified 10 HD-Zip IV members (CsHDZIV1∼10) in tea plant, which are distributed in 8 chromosomes with two segmentally duplicated gene pairs. The exon number of CsHDZIVs ranges from 9 to 11, with most genes having two conserved motifs in the 3’UTR. CsHDZIV1∼9 have almost identical motifs and tertiary structures. The promoter region of CsHDZIVs was predicted to harbor light, phytohormone, stress, growth and developmental elements. 9 CsHDZIVs are divided into 5 subfamilies and located in the nucleus. They exhibited expression trends across 12 tea plant tissues and responded to PEG, cold, NaCl and shading treatments. CsHDZIV3 overexpression partially and completely restored trichome development defects of gl2 leaves and stems, respectively. CsHDZIV3 showed histochemical staining in Arabidopsis trichomes and protein interaction with itself. It might regulate trichome development through forming homodimers. These findings contribute to functional study of tea plant HD-Zip IV and provide guiding significance for the investigation of trichome development and the improvement of trichome traits.

AT-hook motif nuclear localized transcription factors function redundantly in promoting root growth through modulation of redox homeostasis.

Maintaining an optimal redox status is essential for plant growth and development, particularly when the plants are under stress. AT-hook motif nuclear localized (AHL) proteins are evolutionarily conserved transcription factors in plants. Much of our understanding about this gene family has been derived from studies on clade A members. To elucidate the functions of clade B genes, we first analyzed their spatial expression patterns using transgenic plants expressing a nuclear localized GFP under the control of their promoter sequences. AHL1, 2, 6, 7, and 10 were further functionally characterized owing to their high expression in the root apical meristem. Through mutant analyses and transgenic studies, we showed that these genes have the ability to promote root growth. Using yeast one-hybrid and dual luciferase assays, we demonstrated that AHL1, 2, 6, 7, and 10 are transcription regulators and this activity is required for their roles in root growth. Although mutants for these genes did not showed obvious defects in root growth, transgenic plants expressing their fusion proteins with the SRDX repressor motif exhibited a short-root phenotype. Through transcriptome analysis, histochemical staining and molecular genetics experiments, we found that AHL10 maintains redox homeostasis via direct regulation of glutathione transferase (GST) genes. When the transcript level of GSTF2, a top-ranked target of AHL10, was reduced by RNAi, the short-root phenotype in the AHL10-SRDX expressing plant was largely rescued. These results together suggest that AHL genes function redundantly in promoting root growth through direct regulation of redox homeostasis.

Study of the Histological Features of the Stroma of High-Grade Gliomas Depending on the Status of the Mutation in the IDH1 Gene

High-grade gliomas are known for their aggressive nature and resistance to therapy. One characteristic feature of these tumors is the lack of a clear border between the tumor and normal brain tissue. Previous studies have shown that as gliomas dedifferentiate, the extracellular matrix (ECM) undergoes changes in its composition and architecture. This is due to increased production and overexpression of ECM components such as hyaluronic acid, fibulin-3, and collagen. However, it is not yet known what specific changes occur in the stroma of high-grade gliomas depending on the v in the IDH1 gene. In our study, we examined tumor tissue samples from 31 patients, 10 of whom had verified IDH-mutant astrocytoma (grade 4) and 21 had IDH-wildtype glioblastoma (grade 4). The presence or absence of mutations in the IDH1/2 genes was determined in all patients using immunohistochemistry (IHC) and polymerase chain reaction (PCR). To assess stromal changes, we used histochemical staining with Alcian blue and Mallory trichrome. Our results showed significant differences between the two groups according to Student’s t-test (p < 0.05) for all stainings. The presence of mucus formation, collagen formation, and expression of vimentin by tumor cells in the stroma of IDH-wildtype grade 4 glioblastoma indicates an active epithelial-mesenchymal transition and changes in the extracellular matrix. These findings may explain the more unfavorable prognosis in patients with glioblastomas and could potentially serve as a therapeutic target in the complex treatment of malignant gliomas.

Transplantation of miR-145a-5p modified M2 type microglia promotes the tissue repair of spinal cord injury in mice

BackgroundThe traumatic spinal cord injury (SCI) can cause immediate multi-faceted function loss or paralysis. Microglia, as one of tissue resident macrophages, has been reported to play a critical role in regulating inflammation response during SCI processes. And transplantation with M2 microglia into SCI mice promotes recovery of motor function. However, the M2 microglia can be easily re-educated and changed their phenotype due to the stimuli of tissue microenvironment. This study aimed to find a way to maintain the function of M2 microglia, which could exert an anti-inflammatory and pro-repair role, and further promote the repair of spinal cord injury.MethodsTo establish a standard murine spinal cord clip compression model using Dumont tying forceps. Using FACS, to sort microglia from C57BL/6 mice or CX3CR1GFP mice, and further culture them in vitro with different macrophage polarized medium. Also, to isolate primary microglia using density gradient centrifugation with the neonatal mice. To transfect miR-145a-5p into M2 microglia by Lipofectamine2000, and inject miR-145a-5p modified M2 microglia into the lesion sites of spinal cord for cell transplanted therapy. To evaluate the recovery of motor function in SCI mice through behavior analysis, immunofluorescence or histochemistry staining, Western blot and qRT-PCR detection. Application of reporter assay and molecular biology experiments to reveal the mechanism of miR-145a-5p modified M2 microglia therapy on SCI mice.ResultsWith in vitro experiments, we found that miR-145a-5p was highly expressed in M2 microglia, and miR-145a-5p overexpression could suppress M1 while promote M2 microglia polarization. And then delivery of miR-145a-5p overexpressed M2 microglia into the injured spinal cord area significantly accelerated locomotive recovery as well as prevented glia scar formation and neuron damage in mice, which was even better than M2 microglia transplantation. Further mechanisms showed that overexpressed miR-145a-5p in microglia inhibited the inflammatory response and maintained M2 macrophage phenotype by targeting TLR4/NF-κB signaling.ConclusionsThese findings indicate that transplantation of miR-145a-5p modified M2 microglia has more therapeutic potential for SCI than M2 microglia transplantation from epigenetic perspective.Graphical

Different Sources of Bone Marrow Mesenchymal Stem Cells: A Comparison of Subchondral, Mandibular, and Tibia Bone-derived Mesenchymal Stem Cells.

Stem cell properties vary considerably based on the source and tissue site of mesenchymal stem cells (MSCs). The mandibular condyle is a unique kind of craniofacial bone with a special structure and a relatively high remodeling rate. MSCs here may also be unique to address specific physical needs. The aim of this study was to compare the proliferation and multidirectional differentiation potential among MSCs derived from the tibia (TMSCs), mandibular ramus marrow (MMSCs), and condylar subchondral bone (SMSCs) of rats in vitro. Cell proliferation and migration were assessed by CCK-8, laser confocal, and cell scratch assays. Histochemical staining and real-time PCR were used to evaluate the multidirectional differentiation potential and DNA methylation and histone deacetylation levels. The proliferation rate and self-renewal capacity of SMSCs were significantly higher than those of MMSCs and TMSCs. Moreover, SMSCs possessed significantly higher mineralization and osteogenic differentiation potential. Dnmt2, Dnmt3b, Hdac6, Hdac7, Hdac9, and Hdac10 may be instrumental in the osteogenesis of SMSCs. In addition, SMSCs are distinct from MMSCs and TMSCs with lower adipogenic differentiation and chondrogenic differentiation potential. The multidirectional differentiation capacities of TMSCs were exactly the opposite of those of SMSCs, and the results of MMSCs were intermediate. This research offers a new paradigm in which SMSCs could be a useful source of stem cells for further application in stem cell-based medical therapies due to their strong cell renewal and osteogenic capacity.

Embryology of a Lady’s Slipper Orchid, Paphiopedilum spicerianum and Cytokinin Requirements for Seed Germination and Protocorm Development

In this study, we document the primary structural changes that occur during the seed development of Paphiopedilum spicerianum (Rchb.f.) Pfitzer, an endangered species with high horticultural value. Within a defined timeline, our results offer insights into the connection between these structural changes in seeds and their germination percentage. The optimum germination was recorded for immature seeds collected at 180 to 210 days after pollination (DAP), during which the embryos are in the late globular stage and the suspensor begins to degenerate. As seeds continued to mature by 240 DAP, there was a gradual decline in germination. Histochemical staining of mature seeds reveals that only the inner seedcoat and the surface of the embryo exhibit positive reactions to the Nile red stain, suggesting a relatively weak coat-imposed dormancy. This weaker dormancy may contribute to the higher germination observed in mature seeds of P. spicerianum compared with other challenging-to-germinate species. Of the cytokinins examined, 6-(γ,γ-dimethylallylamino)purine (2iP), kinetin (KN), and 6-benzylaminopurine (BA) exhibited a stimulating effect on germination, concurrently enhancing the formation of amorphous protocorms.

Unveiling the Therapeutic Potential of Kelulut (Stingless Bee) Honey in Alzheimer's Disease: Findings from a Rat Model Study.

Alzheimer's disease (AD) poses a major worldwide health challenge because of its profound impact on cognitive abilities and overall well-being. Despite extensive research and numerous clinical trials, therapeutic options remain limited. Our study aimed to investigate the potential of Kelulut honey (KH) as a novel therapeutic agent for addressing the multifactorial pathology of AD. We tried to evaluate the disease-attenuating and neuroprotective potential of KH in the intrahippocampally induced AD rat model by utilizing histochemistry and enzyme-linked immunosorbent assay (ELISA) studies. A total of 26 male Sprague Dawley rats weighing ~280-380 g were randomly divided into three groups: Control, AD-induced (Aβ), and AD-induced and treated with KH (Aβ+KH). The latter two groups underwent stereotaxic surgery, where 6.25 µg of amyloid β1-42 peptides were injected intrahippocampally. One-week post-surgery, KH was administered to the treatment group at a dose of 1 g/kg body weight for a period of four weeks, after which the rats went through behavior tests. After completion of behavior analysis, the rats were sacrificed, and the brains were processed for histochemistry and ELISA studies. The open field test analysis demonstrated that KH improved the locomotion of Aβ+KH compared to Aβ (p = 0.0013). In comparison, the Morris water maze did not show any nootropic effects on cognition with a paradoxical increase in time spent in the target quadrant by the Aβ group (p = 0.029). Histochemical staining showed markedly increased Congo-red-stained amyloid plaques, which were significantly reduced in dentate gyrus of Aβ+KH compared to Aβ (p < 0.05). Moreover, significantly higher apoptosis was seen in the Aβ group compared to Aβ+KH (p < 0.01) and control groups (p < 0.001). Furthermore, the ELISA studies deduced more phosphorylated tau in the diseased group compared to Aβ+KH (p = 0.038) and controls (p = 0.016). These findings suggest that KH consumption for twenty-eight days has the potential to attenuate the pathological burden of disease while exerting neuroprotective effects in rodent models of AD.

Seasonal variation of vessel pits in sapwood: microscopical analyses of the morphology and chemical components of pit membrane encrustations in Fraxinus mandschurica.

Pit pairs and their filter-like partition, i.e. pit membranes, play important roles as water pathways, barriers and regulators in the water-conducting system of angiosperms. In Fraxinus species, the intervessel and vessel-parenchyma pit membranes in sapwood are normally encrusted during winter. Although these encrustations inevitably influence the performance of pits, their properties and functions remain unclear. This study aimed to reveal the morphological and chemical characteristics of encrustations in F. mandshurica in order to deepen understanding of the seasonal encrustation of pit membranes. Seasonal and positional variations in the presence and morphology of encrustations were examined by field-emission scanning electron microscopy (FE-SEM). Cryo-FE-SEM for freeze-fixed greenwood samples was conducted to clarify whether encrustations were present in living trees. Chemical components were examined by histochemical staining using light and electron microscopy, immunofluorescence labelling and ultraviolet microspectroscopy. Encrustations began to deposit in autumn before leaf senescence and disappeared in spring before bud flushing. They infiltrated within the pit membranes, which suggested that they severely limit the permeation of pits. The encrustations differed in morphology among positions: they entirely filled the pit chambers in latewood, while they covered the pit membranes in earlywood. The encrustations were similarly observed in the samples that were freeze-fixed immediately after collection, indicating that they are present in living trees. The encrustations contained polysaccharides, including xyloglucan and homogalacturonan, and phenolic compounds, possibly including flavonoids and coumarins. These chemical components were also detected in droplets found in the latewood vessels with the encrustations, suggesting that the materials constituting encrustations were supplied through the vessel lumens. Encrustations undoubtedly cover the pit membranes in living F. mandshurica trees in winter and their morphology and chemical composition indicate that they are impermeable, have positional differences in function and are characterized by elaborate deposition/removal processes.

Intramuscular administration of fractalkine modulates mitochondrial properties and promotes fast glycolytic phenotype.

A newly categorized myokine called fractalkine (CX3CL1) has been associated with divergent conditions such as obesity, tissue inflammation, and exercise. CX3CL1 works through specific membrane-bound receptors (CX3CR1) found in various tissues including skeletal muscles. Studies indicate CX3CL1 induces muscles to uptake energy substrates thereby improving glucose utilization and countering diabetes. Here, we tested if the administration of purified CX3CL1 directly into mice skeletal muscles affects its histoarchitecture, mitochondrial activity, and expression of metabolic proteins. We analyzed four muscles: two upper-limb (quadriceps, hamstrings) and two lower-limb (tibialis anterior, gastrocnemius), contralateral leg muscles were taken as controls. The effects of CX3CL1 treatment on histoarchitecture, mitochondrial activity, and expression of metabolic proteins in muscles were characterized. We used histochemical staining succinate dehydrogenase (SDH)/cytochrome c oxidase (COX), myosin ATPase, alkaline phosphatase (ALP) to evaluate the mitochondrial activity, fiber types, and vascularization in the muscles, respectively. Western blotting was used to evaluate the expression of proteins associated with mitochondrial metabolism (OXPHOS), glycolysis, and vascularization. Overall, this study indicates CX3CL1 primarily modulates mitochondrial metabolism and shifts substrate preference toward glucose in the skeletal muscle. Evidence also supports that CX3CL1 stimulates the relative composition of fast fiber types, influencing selection of energy substrates in the skeletal muscle.

Transcription factor TaNF-YB2 interacts with partners TaNF-YA7/YC7 and transcriptionally activates distinct stress-defensive genes to modulate drought tolerance in T. Aestivum

BackgroundDrought stress limits significantly the crop productivity. However, plants have evolved various strategies to cope with the drought conditions by adopting complex molecular, biochemical, and physiological mechanisms. Members of the nuclear factor Y (NF-Y) transcription factor (TF) family constitute one of the largest TF classes and are involved in plant responses to abiotic stresses.ResultsTaNF-YB2, a NY-YB subfamily gene in T. aestivum, was characterized in this study focusing on its role in mediating plant adaptation to drought stress. Yeast two-hybrid (Y-2 H), biomolecular fluoresence complementation (BiFC), and Co-immunoprecipitation (Co-IP) assays indicated that TaNF-YB2 interacts with the NF-YA member TaNF-YA7 and NF-YC family member TaNF-YC7, which constitutes a heterotrimer TaNF-YB2/TaNF-YA7/TaNF-YC7. The TaNF-YB2 transcripts are induced in roots and aerial tissues upon drought signaling; GUS histochemical staining analysis demonstrated the roles of cis-regulatory elements ABRE and MYB situated in TaNF-YB2 promoter to contribute to target gene response to drought. Transgene analysis on TaNF-YB2 confirmed its functions in regulating drought adaptation via modulating stomata movement, osmolyte biosynthesis, and reactive oxygen species (ROS) homeostasis. TaNF-YB2 possessed the abilities in transcriptionally activating TaP5CS2, the P5CS family gene involving proline biosynthesis and TaSOD1, TaCAT5, and TaPOD5, the genes encoding antioxidant enzymes. Positive correlations were found between yield and the TaNF-YB2 transcripts in a core panel constituting 45 wheat cultivars under drought condition, in which two types of major haplotypes including TaNF-YB2-Hap1 and -Hap2 were included, with the former conferring more TaNF-YB2 transcripts and stronger plant drought tolerance.ConclusionsTaNF-YB2 is transcriptional response to drought stress. It is an essential regulator in mediating plant drought adaptation by modulating the physiological processes associated with stomatal movement, osmolyte biosynthesis, and reactive oxygen species (ROS) homeostasis, depending on its role in transcriptionally regulating stress response genes. Our research deepens the understanding of plant drought stress underlying NF-Y TF family and provides gene resource in efforts for molecular breeding the drought-tolerant cultivars in T. aestivum.

Effect of Two Forms of Tenofovir on Duodenal Enterocytes - a Hypothesis for Different Effect of TDF and TAF on Body Weight and Plasma Lipids.

Tenofovir disoproxil fumarate (TDF) compared to tenofovir alafenamide (TAF) leads to lower body weight and plasma lipids by an unknown mechanism. We hypothesize that TDF, when absorbed, may damage enterocytes of the proximal duodenum, leading to reduced absorption of nutrients. People living with HIV, without significant gastrointestinal symptoms, receiving TDF (n=12) or TAF (n=12) containing regimen underwent esophagogastroduodenoscopies with duodenal biopsies. Plasma/serum concentrations of nutrients absorbed from proximal duodenum and serum intestinal fatty-acid-binding protein (I-FABP), a marker of enterocyte damage, were measured. COX/SDH histochemical staining and electron microscopy (EM) were conducted to evaluate mitochondria. Five patients in TDF (celiac disease (excluded from further analyses), helicobacter gastritis, and three esophagitis) and two in TAF group (two esophagitis) had a pathological finding in esophagogastroduodenoscopy. Villi were flatter (337 (59) vs. 397 (42) μm, p=0.016), crypts non-significantly deeper (200 (46) vs. 176 (27) μm, p=0.2), and villus to crypt ratio lower (1.5 (0.42) vs. 2.5 (0.51), p=0.009) in TDF vs. TAF group. I-FABP concentration was higher in TDF vs. TAF group (3.0 (1.07) vs. 1.8 (0.53) ng/ml, p=0.003). TDF group had numerically but not statistically significantly lower concentrations of folate, vitamins A, B1, D, and E. COX/SDH staining showed signs of mitochondrial damage in 10 participants in TDF and 11 in TAF group. EM studies showed similar mitochondrial damage in both groups. Duodenal villous alterations may explain TDF-associated decrease in body weight and plasma lipids. Larger studies are needed to evaluate concentrations of nutrients absorbed from duodenum among TDF users.

Clinical and pathological characteristics of OPDM4 patients in advanced disease.

Oculopharyngodistal myopathy type 4 (OPDM4) arises from a CGG repeat expansion in the 5' UTR of the RILPL1 gene. Reported cases of OPDM4 have been limited. The aim of this study was to investigate the clinical and myopathological characteristics of OPDM4 patients with advanced disease. We assessed a total of 8 affected and 12 unaffected individuals in an OPDM4 family with autosomal dominant inheritance. Muscle biopsy tissue from the proband underwent histological, enzyme histochemical, and immunohistochemical stains, and electron microscopy analysis. Whole exome sequencing and repeat primer PCR (RP-PCR) were conducted to investigate underlying variants. OPDM4 patients displayed a progressive disease course. Most experienced lower limb weakness and diminished walking ability in their 20s and 30s, followed by ptosis, ophthalmoplegia, swallowing difficulties, and dysarthria in their 30s to 50s, By their 50s to 70s, they became non-ambulatory. Muscle magnetic resonance imaging (MRI) of the proband in advanced disease revealed severe fatty infiltration of pelvic girdle and lower limb muscles. Biopsied muscle tissue exhibited advanced changes typified by adipose connective tissue replacement and the presence of multiple eosinophilic and p62-positive intranuclear inclusions. Immunopositivity for the intranuclear inclusions was observed with anti-glycine antibody and laboratory-made polyA-R1 antibody. RP-PCR unveiled an abnormal CGG repeat expansion in the 5' UTR of the RILPL1 gene. The clinical and radiological features in this family broaden the phenotypic spectrum of OPDM4. The presence of intranuclear inclusions in the proliferative adipose connective tissues of muscle biopsy specimens holds diagnostic significance for OPDM4 in advanced disease.

Syringaresinol Attenuates α-Melanocyte-Stimulating Hormone-Induced Reactive Oxygen Species Generation and Melanogenesis.

Ginseng has been utilized for centuries in both the medicinal and cosmetic realms. Recent studies have actively investigated the biological activity of ginseng berry and its constituents. (+)-Syringaresinol [(+)-SYR], an active component of ginseng berry, has been demonstrated to have beneficial effects on the skin, but its potential impact on skin pigmentation has not been fully explored. Here, the antioxidant and anti-pigmentary activity of (+)-SYR were evaluated in B16F10 murine melanoma cells and in an artificial human pigmented skin model, Melanoderm™. A real-time PCR, Western blotting, immunofluorescence staining, and histochemistry staining were conducted to confirm the effects of (+)-SYR on pigmentation. (+)-SYR reduced melanogenesis and dendrite elongation in α-melanocyte-stimulating hormone (α-MSH)-primed B16F10 cells with low cytotoxicity. (+)-SYR suppressed the expression of melanogenic genes, namely tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). Notably, (+)-SYR attenuated α-MSH-induced cytosolic and mitochondrial reactive oxygen species (ROS) generation, which was attributable at least in part to the suppression of NADPH oxidase-4 (NOX 4) expression. Finally, the brightening activities of (+)-SYR were verified using Melanoderm™, underscoring the potential of ginseng berry and (+)-SYR as functional ingredients in skin-brightening cosmetics.

Promoter of Cassava MeAHL31 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis.

The AT-hook motif nuclear-localized (AHL) family is pivotal for the abiotic stress response in plants. However, the function of the cassava AHL genes has not been elucidated. Promoters, as important regulatory elements of gene expression, play a crucial role in stress resistance. In this study, the promoter of the cassava MeAHL31 gene was cloned. The MeAHL31 protein was localized to the cytoplasm and the nucleus. qRT-PCR analysis revealed that the MeAHL31 gene was expressed in almost all tissues tested, and the expression in tuber roots was 321.3 times higher than that in petioles. Promoter analysis showed that the MeAHL31 promoter contains drought, methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellin (GA) cis-acting elements. Expression analysis indicated that the MeAHL31 gene is dramatically affected by treatments with salt, drought, MeJA, ABA, and GA3. Histochemical staining in the proMeAHL31-GUS transgenic Arabidopsis corroborated that the GUS staining was found in most tissues and organs, excluding seeds. Beta-glucuronidase (GUS) activity assays showed that the activities in the proMeAHL31-GUS transgenic Arabidopsis were enhanced by different concentrations of NaCl, mannitol (for simulating drought), and MeJA treatments. The integrated findings suggest that the MeAHL31 promoter responds to the abiotic stresses of salt and drought, and its activity is regulated by the MeJA hormone signal.

Mechanistic study of the effect of flexible fixation and load-bearing stress environment on fracture healing and shaping.

The biomechanical environment created by suture-button fixation Latarjet is conducive to the healing and shaping of the transplanted coracoid, but its mechanism remains unclear. The latest research has found that the absence of stem cell chemokine (CXCL12) impeded bone regeneration in Sonic Hedgehog (SHH)-deficient animals. However, whether the biomechanical environment affects SHH and CXCL12 function has not been studied. Rat fracture models were constructed to simulate stress environments under non-load-bearing and load-bearing conditions. The fracture healing and shaping, as well as the expression levels of SHH and CXCL12, were assessed through gross viewing, micro-computed tomography (micro-CT), and histochemical staining. Under flexible fixation, the relative bone volume (BV/TV) of rats exposed to the load-bearing stress environment was significantly higher than that of rats under a non-load-bearing stress environment (p ≤ 0.05). Adverse bone shaping was not observed in rats subjected to flexible fixation. The levels of SHH and CXCL12 in load-bearing rats exhibited significant elevation (p ≤ 0.05). Under a load-bearing stress environment, no significant difference was observed in the BV/TV between the flexible fixation group and the rigid fixation group (p ≥ 0.05), but there was excessive hyperplasia of the fracture callus in the rigid fixation group. The levels of SHH and CXCL12 in rats subjected to rigid fixation were significantly elevated (p ≤ 0.05). Flexible fixation and load-bearing stress environment may contribute to bone healing and shaping by influencing the levels of SHH and CXCL12, suggested that this mechanism may be relevant to the healing and shaping of the transplanted coracoid after suture-button fixation Latarjet.

Regulating Tissue Growth Factors for Healing With Etherified Carboxymethylcellulose Matrix.

Etherified Carboxymethylcellulose Matrix (eCMC) is a revolutionary application of carboxymethylcellulose (CMC) in wound care, known for its potential in hemostasis and tissue regeneration. This study aims to investigate the mechanism of eCMC in tissue healing by establishing a rat burn model and administering eCMC as a treatment. The objective is to analyze cytokines and inflammatory mediators using a Cytokine Array and histochemical staining to understand the effects of eCMC on tissue regeneration. A rat burn model was created, and eCMC was applied as a treatment. Tissue samples were collected at multiple time points to assess the expression of cytokines and inflammatory mediators using a Cytokine Array. In addition, histochemical staining was performed to evaluate tissue regeneration factors. eCMC induced the expression of endogenous cytokines, particularly vascular epithelial growth factor and platelet-derived growth factor, while inhibiting inflammatory cytokines such as CINC-1, CINC-2, and MMP-8. This dual action facilitated wound healing and mitigated the risk of infection. eCMC demonstrates promising potential for enhancing skin regeneration. Further research is warranted to delve into the precise mechanism of eCMC's cytokine regulation. In vitro and in vivo studies should be conducted to comprehensively investigate the therapeutic capabilities of eCMC in wound healing.

Morphological examination of the visual system and orbital region in the red panda (Ailurus fulgens fulgens)

ObjectivesThe red panda is currently the only surviving member of the Ailuridae family in the Caniformia suborder. In this study, we provide data on anatomical, morphometric, histological and histochemical examination of the orbital region, eyelids, orbital gland, and eye tunics in two adult males Ailurus fulgens fulgens from the Wroclaw Zoological Garden, Poland.MethodsThe study involved morphometric analysis of the eyeball and selected accessory organs of the eye, along with analysis of the bony orbit, including its morphometry, macroscopic, and microscopic evaluation. Microscopic evaluation encompassed histological and histochemical staining, with the former involving hematoxylin & eosin (H&E), Movat pentachrome, picro-Mallory trichrome, Fontana-Masson, and the latter including PAS, AB pH 1.0, AB pH 2.5; AB pH 2.5/PAS, and HDI.ResultsThe upper (UE) and lower (LE) eyelids presented well-developed tarsal glands, sebaceous glands, and a characteristic simple alveolar gland (producing a mucous secretion). The palpebral part of the lacrimal gland was present. A single lymphoid follicle was observed only in the upper eyelids. The superficial gland of the third eyelid (SGTE) was a multilobar acinar complex that produces mucous secretion and is contained within the interlobular ducts of numerous aggregates of lymph nodes. The third eyelid (TE) was T-shaped and composed of hyaline tissue, containing CALT. The lacrimal gland (LG) also revealed a multilobar acinar complex that produced mucous secretion, with a single lymphoid follicle. The cornea consisted of 4 layers, as Bowman’s membrane was absent. The Vogt palisades composed of 7–10 layers of epithelial cells were demonstrated. The pupil was horizontally ovoid at rest (post-mortem). The sphincter pupil and the dilator pupil were well developed. Macroscopically, the tapetum lucidum appeared as a milky, non-opalescent crescent. Histologically, the choroidal tapetum lucidum cellulosum consisted of 5 to 9 layers of loosely packed oval cells. The retina showed a composition similar to that of terrestrial nocturnal carnivores.ConclusionsThe results of our research indicate that the anatomical features of the eye and orbital region in the red panda share similarities with those described in the Musteloidea clade, as well as the Canidae and Ursidae families.