[Advances of pathological diagnosis and molecular pathology of lung neuroendocrine neoplasms].

The pathological classification and diagnostic criteria for lung neuroendocrine neoplasms (NENs) in the 2021 World Health Organization (WHO) lung tumor classification are similar to the prior classifications. However, the advances on the molecular studies of lung NENs have shown that both small cell lung carcinoma and large cell neuroendocrine carcinoma are highly heterogeneous tumors with neuroendocrine characteristics and can be subclassified based on the features of genomics or transcriptomics, which are valuable in the diagnosis of lung NENs subtypes and patient treatment. In addition, it is necessary to interpret emerging concepts such as "lung neuroendocrine tumor G3" and "histological transformation" from pathological perspectives, as well as to know the novel neuroendocrine biomarkers such as INSM1 and POU2F3. This article summarized the diagnostic changes and the advances of molecular pathology of lung NENs based on the latest WHO classification and molecular research.

[Genetic changes and biological potential of proliferative nodule in congenital pigmented nevus].

Objective: To study the genetic changes and biological potential of proliferative nodule in congenital melanocytic nevus. Methods: Whole-exome sequencing was carried out using the technique of next-generation sequencing (NGS) in order to detect the genomic alterations of two cases of proliferative nodules (PN) in congenital melanocytic nevi (CMN). Twelve cases of CMN and ten cases of malignant melanoma were used as benign and malignant controls, respectively. Mutated genes that possessed statistically significant difference between benign and malignant controls were listed, according to what benign and malignant statuses were classified and clustered. The heatmaps of clustering analyses were depicted using heatmap package. Fluorescence in situ hybridization (FISH) was also used to validate the above results. Results: Eighty-six common somatic gene mutations were detected in two samples of PN. Compared with CMN, PN had 52 more mutated genes. Furthermore, 22 of these 52 mutated genes were also detected in malignant melanoma samples. Two cases of PN fell between benign CMN and malignant melanoma in germline mutation clustering. Both cases of PN were positive in the FISH tests. Conclusions: The genetic changes of PN partially overlap with those of CMN and malignant melanoma. Therefore, although most of the PN manifest as a benign lesion clinically, it may have certain malignant potential at the genetic level, and warrant long-term monitoring and follow-up.

[The correlation study of ground glass opacity and lepidic growth pattern component in stage I lung invasive adenocarcinoma].

Objective: To investigate the correlation between Ground Glass Opacity (GGO) component proportion and quantitative classification of lepidic growth pattern in pathological stage Ⅰpulmonary adenocarcinoma. Methods: Pathological and HRCT data of 183 stage Ⅰ invasive adenocarcinoma patients from January 2005 to December 2012 were retrospectively reviewed. The proportion of GGO was calculated from diameter and volume.The correlation between GGO component proportion and lepidic growth pattern in pathological were analyzed by Spearman correlation. Results: Among 183 patients, the proportion of GGO component calculated by maximum diameter method and three-dimensional computerized quantification was 0.43±0.35 and 0.20±0.18, respectively. The percentage of lepidic growth pattern component using semi-quantitative analysis of pathological sections was 0.29±0.25.The proportion of GGO by diameter and three-dimensional computerized quantification was significantly correlated with the percentage of lepidic growth pattern component (r=0.599, P< 0.001; r=0.620, P<0.001). Conclusions: There was a positive correlation between the content of lepidic growth pattern and the content of GGO in the small adenocarcinoma. Three-dimensional computerized quantification was a better method in preoperational evaluation.

A novel regulatory function for miR-29a in keloid fibrogenesis.

BACKGROUND: A growing body of evidence has shown that microRNA-29 (miR-29) plays a central role in the progression of fibrosis. However, the mechanisms underlying the role of miR-29 in keloid fibrogenesis remain unknown. AIM: To investigate the roles of miR-29 in dermal fibroblasts in the pathogenesis of keloids. METHODS: Primary fibroblasts from 9 patients with keloid and 6 healthy controls (HCs) were cultured and pretreated with transforming growth factor (TGF)-ß1. Next, fibroblasts were transfected with precursor miRNA and anti-miR-29a miRNA. TGF-ß1-associated miR-29 alterations were investigated by quantitative real-time PCR. Collagen I and collagen III protein levels were analysed by western blotting. RESULTS: miR-29a, miR-29b and miR-29c levels were significantly lower in keloid compared with healthy fibroblasts (P < 0.05), and in particular, miR-29a was especially markedly reduced (P < 0.001). Type I and type III collagen mRNA and protein levels were decreased in keloid fibroblasts transfected with pre-miR-29a (P < 0.05), whereas knockdown with anti-miR-29a increased type I and type III collagen mRNA and protein expression (P < 0.05) in the fibroblasts. Interestingly, pretreatment of fibroblasts with TGF-ß1 significantly decreased miR-29a (P < 0.05), whereas miR-29b and miR-29c were reduced to a lesser extent, which was not significant. CONCLUSIONS: These findings show that miR-29a exerts as a novel regulator in the fibrogenesis of keloid, suggesting that miR-29a might be a novel marker for keloid.

Altered circulating endothelial progenitor cells in patients with keloid.

Evidence has suggested that vascular endothelial growth factor (VEGF), a crucial growth factor in regulating endothelial progenitor cells (EPCs), plays a central role in keloid formation. However, the levels of circulating EPCs in patients with keloid have not yet been explored. The aim of this study was to determine the number of circulating EPCs in patients with keloid. Circulating EPCs (defined as CD45- CD34+CD133+VEGFR2+cells) and VEGF levels from 39 patients with keloid and 22 healthy controls (HCs) were assessed by flow cytometry and ELISA, respectively. EPCs were detectable in the peripheral blood of patients with keloid. The number of circulating EPCs and the levels of plasma VEGF were significantly higher in patients with keloid than in HCs. However, no correlation was found between the number of circulating EPCs and the serum VEGF levels. This study provides the first evidence that EPCs are increased in the peripheral blood of patients with keloid. Understanding the roles of EPCs in keloid fromation may lead to the development of novel therapeutic strategies for keloid.

Role of the NO/cGMP pathway in postoperative vasodilation in perforator flaps.

BACKGROUND: The nitric oxide (NO)/cyclic guanylyl monophosphate (cGMP) pathway is one of the most important regulators of tissue perfusion. Here, we sought to elucidate the protective effects of the NO/cGMP pathway on the microcirculation of axial pattern skin flaps. MATERIAL AND METHODS: Overall 40 rats were divided into four groups (n = 10 each): group A, sildenafil was administered orally at 10 mg/kg daily; group B, sildenafil citrate (10 mg/kg, oral) and nitro-amino-methyl-L-arginine (L-NAME, intraperitoneal injection), a nitric oxide synthase inhibitor, were administered daily; group C, L-NAME was administered alone; and group D, no drugs were administered. After surgery, the surviving flap area was calculated as a percentage of total flap dimensions using the paper template technique. Angiography and imaging were performed to compare the macrovascular changes of the choke zones in the flaps. Histological examinations were performed to compare the differences in microvascular changes between the two choke zones. RESULTS: A significant improvement of flap survival area and a significant dilation of vessels in both choke zones were found after administration of sildenafil. We also found that the postoperative vasodilation of choke vessels could be altered by inhibition of NO synthase (NOS). Moreover, the vasodilatory effect prolonged by the phosphodiesterase 5 inhibitor sildenafil was attenuated after administration of L-NAME. L-NAME significantly reversed the protection afforded by sildenafil. CONCLUSIONS: Targeting the NO/cGMP pathway can dilate vessels along the axis of the flap, including the choke vessels, thus augmenting flap viability. Therefore, targeting of this pathway may have therapeutic applications.

Air-assisted intranasal instillation enhances adenoviral delivery to the olfactory epithelium and respiratory tract.

Intranasal instillation is used to deliver adenoviral vectors to the olfactory epithelium and respiratory tract. The success of this approach, however, has been tempered by inconsistent infectivity in both the epithelium and lungs. Infection of the epithelium may be hampered in part by the convoluted structure of the cavity, the presence of mucus or poor airflow in the posterior cavity. Delivery of adenovirus to the lungs can be uneven in the various lobes and distal bronchioles may be poorly infected. Current approaches to circumvent these issues rely principally on intubation or intratracheal instillation. Here we describe a technique that significantly improves adenoviral infectivity rates without requiring surgical intervention. We use compressed air to increase circulation of instilled adenovirus, resulting in enhanced infection in both the epithelium and lungs. This procedure is straightforward, simple to perform and requires no specialized equipment. In the epithelium, neurons and sustentacular cells are both labeled. In the lungs, all lobes can be infected, with penetration to the most distal bronchioles. The use of compressed air will likely also be useful for enhancing the distribution of other, desired agents within the epithelium, central nervous system and respiratory tract.

Genetic investigation of nodal melanocytic nevi in cases of giant congenital melanocytic nevus.

Nodal melanocytic nevi are common incidental findings in lymph nodes that have been removed during sentinel lymph node biopsy for melanoma. They can also occur in the local lymph nodes of the giant congenital nevus (GCN), but very little is known regarding nodal melanocytic nevi in the giant congenital nevus, especially at the genetic level. There are two theories that explain the possible pathogenesis of nodal melanocytic nevi, mechanical transport and arrested migration during embryogenesis. However, there have been few tests of these two theories at the molecular biology level until now. We used whole-exon sequencing to test these two theories at the gene level for the first time. In clonal evolution analysis of patient 1, whose tumor mutation burden (TMB) value was relatively stable, showed that the GCN and nodal nevus had the same initial origin and then diverged into two branches as a result of gene mutations. In contrast, analysis indicated that in the other patient, whose TMB value declined from 68.02/Mb in a GCN to 17.55/Mb in associated nodal nevi, these two samples were from different origins at the beginning, each with its own gene mutation. These results are consistent with the two respective theories at the molecular biological level. We provided the first tests of the two theories of pathogenesis of nodal melanocytic nevi at the gene level, and these findings may provide some clues for further study. In addition, not all nodal nevi should be treated as lymph node metastasis in clinical diagnosis, and we should make a comprehensive assessment and judgment of nodal melanocytic nevi based on morphology, immunological characteristics and fluorescence in situ hybridization (FISH) tests.

Postconditioning Protection Against Myocardiocyte Anoxia/Reoxygenation Injury From Penehyclidine Hydrochloride.

BACKGROUND/AIMS: To investigate the postconditioning protective effect of penehyclidine hydrochloride (PHC) against anoxia/reoxygenation (A/R) injury in H9c2 cells along with the involved mechanism and timing effect. METHODS: We divided H9c2 cells into 7 groups: control group, A/R group and PHC+A/R groups at 0 min, 5 mins, 10 mins, 20 mins, 30 mins, respectively (treated with 0.1 µm/L PHC at 0 min, 5 mins, 10 mins, 20 mins, 30 mins after the reoxygenation procedure began). Cell apoptosis, oxidative stress, intracellular Ca2+ concentration, mitochondrial membrane potential and mitochondrial permeability transition pore (MPTP) opening were explored. Bcl-2, Bax, Cyt C, caspase-3 and caspase-9 levels were measured. RESULTS: A/R significantly increased both cell injury and cell apoptosis. PHC showed postconditioning protective effect by attenuating superoxide production, decreasing Ca2+ overload, restraining MPTP activities, restoring mitochondrial membrane potential, regulating cell apoptosis proteins and modulation of mitochondrial pathway. Earlier administration of PHC offered greater postconditioning protective effect. CONCLUSION: H9c2 cells were protected by PHC from A/R injury regardless of timing of PHC administration (0 min, 5 mins, 10 mins, 20 mins, 30 mins). However, earlier administration of PHC resulted in better PHC postconditioning protection.

Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance.

We used the enhancer detection/GAL4 system in Drosophila to direct increased levels of Fasciclin II (Fas II) expression on motoneuron growth cones and axons and to direct ectopic Fas II expression on other cells they encounter. Four classes of abnormal phenotypes are observed: "bypass" phenotypes, in which axons fail to defasciculate at the choice point where they would normally enter their muscle target region and instead extend past their target; "detour" phenotypes, in which these bypass growth cones enter their muscle target region at a different location; "stall" phenotypes, in which axons that enter their muscle target region fail to defasciculate from one another to probe their muscle targets; and "misroute" phenotypes, in which growth cones are diverted onto abnormal pathways by contact with Fas II-positive cells. These phenotypes show that changes in the pattern and level of Fas II expression can alter growth cone guidance, apparently in part by modulating the ability of these growth cones to respond to other guidance cues.

Targeted neuronal cell ablation in the Drosophila embryo: pathfinding by follower growth cones in the absence of pioneers.

We developed a rapid method that uses diphtheria toxin, the flp recognition target sequences, and the GAL4-UAS activation system, to ablate specific neurons in the Drosophila embryo and to examine the consequences in large numbers of embryos at many time points. We used this method to show that, in the absence of the aCC axon, which pioneers the intersegmental nerve in the PNS, the three U follower axons are delayed and make frequent errors. However, the pathway ultimately forms in most segments. We also ablated the axons that pioneer the first longitudinal pathways within the CNS and observed similar results; the formation of longitudinal pathways is delayed and disorganized in 70% of segments, but these tracts ultimately form in 80% of segments. Thus, pioneers facilitate the development of PNS and CNS axon pathways; in their absence, followers are delayed and make numerous errors. However, pioneers are not absolutely required, as these embryos display a remarkable ability to correct for the loss of the pioneering neurons.

Genetic analysis of Fasciclin II in Drosophila: defasciculation, refasciculation, and altered fasciculation.

The Drosophila neural cell adhesion molecule Fasciclin II (Fas II) is expressed dynamically on a subset of embryonic CNS axons, many of which selectively fasciculate in the vMP2, MP1, and FN3 pathways. Here we show complementary fasII loss-of-function and gain-of-function phenotypes. Loss-of-function fasII mutations lead to the complete or partial defasciculation of all three pathways. Gain-of-function conditions, using a specific control element to direct increased levels of Fas II on the axons in these three pathways, rescue the loss-of-function phenotype. Moreover, the gain-of-function can alter fasciculation by abnormally fusing pathways together, in one case apparently by preventing normal defasciculation. These results define an in vivo function for Fas II as a neuronal recognition molecule that controls one mechanism of growth cone guidance-selective axon fasciculation--and genetically separates this function from other aspects of outgrowth and directional guidance.

Formation of precise connections in the olfactory bulb occurs in the absence of odorant-evoked neuronal activity.

Olfactory neurons expressing the same odorant receptor converge to a small number of glomeruli in the olfactory bulb. In turn, mitral and tufted cells receive and relay this information to higher cortical regions. In other sensory systems, correlated neuronal activity is thought to refine synaptic connections during development. We asked whether the pattern of connections between olfactory sensory axons and mitral cell dendrites is affected when odor-evoked signaling is eliminated in mice lacking functional olfactory cyclic nucleotide-gated (CNG) channels. We demonstrate that olfactory sensory axons converge normally in the CNG channel mutant background. We further show that the pruning of mitral cell dendrites, although slowed during development, is ultimately unperturbed in mutant animals. Thus, the olfactory CNG channel-and by inference correlated neural activity--is not required for generating synaptic specificity in the olfactory bulb.

Functional identification of a goldfish odorant receptor.

The vertebrate olfactory system utilizes odorant receptors to receive and discriminate thousands of different chemical stimuli. An understanding of how these receptors encode information about an odorant's molecular structure requires a characterization of their ligand specificities. We employed an expression cloning strategy to identify a goldfish odorant receptor that is activated by amino acids-potent odorants for fish. Structure-activity analysis indicates that the receptor is preferentially tuned to recognize basic amino acids. The receptor is a member of a multigene family of G protein-coupled receptors, sharing sequence similarities with the calcium sensing, metabotropic glutamate, and V2R class of vomeronasal receptors. The ligand tuning properties of the goldfish amino acid odorant receptor provide information for unraveling the molecular mechanisms underlying olfactory coding.

Bacterial infection promotes DNA hypermethylation.

Maternal oral infection, caused by bacteria such as C. rectus or P. gingivalis, has been implicated as a potential source of placental and fetal infection and inflammatory challenge, which increases the relative risk for pre-term delivery and growth restriction. Intra-uterine growth restriction has also been reported in various animal models infected with oral organisms. Analyzing placental tissues of infected growth-restricted mice, we found down-regulation of the imprinted Igf2 gene. Epigenetic modification of imprinted genes via changes in DNA methylation plays a critical role in fetal growth and development programming. Here, we assessed whether C. rectus infection mediates changes in the murine placenta Igf2 methylation patterns. We found that infection induced hypermethylation in the promoter region-P0 of the Igf2 gene. This novel finding, correlating infection with epigenetic alterations, provides a mechanism linking environmental signals to placental phenotype, with consequences for development.