Single cell deciphering of progression trajectories of the tumor ecosystem in head and neck cancer.

Head and neck squamous cell carcinoma is the sixth most common cancer worldwide and has high heterogeneity and unsatisfactory outcomes. To better characterize the tumor progression trajectory, we perform single-cell RNA sequencing of normal tissue, precancerous tissue, early-stage, advanced-stage cancer tissue, lymph node, and recurrent tumors tissue samples. We identify the transcriptional development trajectory of malignant epithelial cells and a tumorigenic epithelial subcluster regulated by TFDP1. Furthermore, we find that the infiltration of POSTN+ fibroblasts and SPP1+ macrophages gradually increases with tumor progression; their interaction or interaction with malignant cells also gradually increase to shape the desmoplastic microenvironment and reprogram malignant cells to promote tumor progression. Additionally, we demonstrate that during lymph node metastasis, exhausted CD8+ T cells with high CXCL13 expression strongly interact with tumor cells to acquire more aggressive phenotypes of extranodal expansion. Finally, we delineate the distinct features of malignant epithelial cells in primary and recurrent tumors, providing a theoretical foundation for the precise selection of targeted therapy for tumors at different stages. In summary, the current study offers a comprehensive landscape and deep insight into epithelial and microenvironmental reprogramming throughout initiation, progression, lymph node metastasis and recurrence of head and neck squamous cell carcinoma.

LncRNA MIR4435-2HG contributes into colorectal cancer development and predicts poor prognosis.

OBJECTIVE: Some studies have confirmed that long non-coding ribonucleic acids (lncRNAs) played a vital role in the pathophysiology of various diseases, especially in oncogenesis and progression of tumors. Dysexpressed lncRNAs regulate different cellular processes, including proliferation, invasion, and apoptosis. The aim of this study was to explore the clinical significance and function of lncRNA MIR4435-2HG in colorectal cancer. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to detect the expression of lncRNA MIR4435-2HG. The Kaplan-Meier method was used to evaluate the overall survival and disease-free survival of patients with colorectal cancer. The cell proliferation was measured by Methyl thiazolyl tetrazolium (MTT) assay. The cell apoptotic rate was measured via flow cytometry method. RESULTS: LncRNA MIR4435-2HG is a novel cancer-related lncRNA that was recently found to exhibit high expression in colorectal cancer. The dysregulation of lncRNA miR4435-2HG was significantly related to the tumor size (p<0.001), lymph node metastasis (p<0.001), and tumor node metastasis (TNM) staging (p=0.022). The patients with higher expression of lncRNA miR4435-2HG showed worse prognosis than those with low expression of lncRNA miR4435-2HG group. Besides, the downregulated lncRNA miR4435-2HG expression could repress cell proliferation and enhance cell apoptosis. CONCLUSIONS: LncRNA MIR4435-2HG functions as an oncogene that promotes colorectal cancer progression, and likely represents a biomarker or therapeutic target of colorectal cancer.

Colorectal anastomosis after laparoscopic extended left colectomy: techniques and outcome.

AIM: After extended left colectomy, traditional colorectal anastomosis is often not feasible because of insufficient length of the remaining colon to perform a tension-free anastomosis. Total colectomy with ileorectal anastomosis could be an alternative but this can lead to unsatisfactory quality of life. Trans-mesenteric colorectal anastomosis or inverted right colonic transposition (the so-called Deloyers procedure) are two possible solutions for creating a tension-free colorectal anastomosis after extended left colectomy. Few studies have reported their results of these two techniques and mostly via laparotomy. The aim of this study was to describe the trans-mesenteric colorectal anastomosis and the inverted right colonic transposition procedure via a laparoscopic approach and report the outcome in a series of 13 consecutive patients. METHOD: This was retrospective chart review of laparoscopic colorectal surgery with trans-mesenteric colorectal anastomosis or the inverted right colonic transposition procedure from January 2015 up to 2019. An accompanying video demonstrates these two techniques. RESULTS: Thirteen consecutive patients underwent either a laparoscopic trans-mesenteric colorectal anastomosis (n = 9) or an inverted right colonic transposition procedure (n = 4). One patient had intra-operative presacral bleeding that was stopped successfully without conversion. Two patients had a postoperative intra-abdominal abscess, but no anastomotic complications were recorded. The median number of bowel movements per day after 6 months was 2 (range 2-5). CONCLUSIONS: Trans-mesenteric colorectal anastomosis or the inverted right colonic transposition procedure is feasible laparoscopically. The now well-established classical advantages of the laparoscopic approach are associated with good functional outcome after these procedures.

MicroRNA-125a regulates proliferation and apoptosis of acute myeloid leukemia through targeting NF-κB pathway.

OBJECTIVE: To elucidate the influence of microRNA-125a on the biological behaviors of acute myeloid leukemia (AML) cells. MATERIALS AND METHODS: MicroRNA-125a mimic and negative control (NC) were constructed and transfected into AML cell line HL60, respectively. Cell viability of HL60 cells transfected with microRNA-125a mimic or NC was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Regulatory effects of microRNA-125a on enzyme activities of B-cell lymphoma-2 (Bcl-2), Bcl-xl, caspase-3, and caspase-9 in HL60 cells were quantified by a spectrophotometry. Changes in apoptosis and invasion of HL60 cells overexpressing microRNA-125a were detected by flow cytometry and transwell assay, respectively. Protein levels of cell cycle genes (cyclin B, cdc-2, mdm-2), pro-apoptotic gene p53 and anti-apoptotic gene Bcl-2 in HL60 cells transfected with microRNA-125a mimic or NC were assessed by Western blot. Finally, the mRNA levels of Bax, caspase-8, nuclear factor-κB (NF-κB), and c-myc in HL60 cells with microRNA-125a overexpression were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). RESULTS: MicroRNA-125a expression remarkably increased by transfection of microRNA-125a mimic into HL60 cells, suggesting its sufficient transfection efficacy. MTT assay revealed an inhibited viability after microRNA-125a overexpression. Transfection of microRNA-125a mimic markedly enhanced enzyme activities of caspase-3 and caspase-9, but reduced activities of Bcl-2 and Bcl-xl in HL60 cells than controls (p<0.05). Moreover, microRNA-125a overexpression elevated apoptotic rate as FCM data indicated. Transwell assay demonstrated a decrease in the invasive rate of HL60 cells overexpressing microRNA-125a. Western blot analyses revealed that cell cycle genes all downregulated by transfection of microRNA-125a mimic in HL60 cells. The protein level of p53 upregulated and Bcl-2 downregulated in HL60 cells overexpressing microRNA-125a (p<0.05). Furthermore, mRNA levels of pro-apoptotic genes Bax and caspase-8 were enhanced after microRNA-125a overexpression, while mRNA levels of NF-κB and c-myc were reduced (p<0.05). CONCLUSIONS: MicroRNA-125a inhibits proliferative and invasive potentials, arrests the cell cycle in the G2/M phase of AML cells by regulating the NF-κB pathway.

Regulation of c-fos expression by the dopamine D1-D2 receptor heteromer.

The dopamine D1 and D2 receptors form the D1-D2 receptor heteromer in a subset of neurons and couple to the Gq protein to regulate intracellular calcium signaling. In the present study the effect of D1-D2 heteromer activation and disruption on neuronal activation in the rat brain was mapped. This was accomplished using the dopamine agonist SKF 83959 to activate the D1-D2 heteromer in combination with a TAT-D1 disrupting peptide we developed, and which has been shown to disrupt the D1/D2 receptor interaction and antagonize D1-D2 heteromer-induced cell signaling and behavior. Acute SKF 83959 administration to rats induced significant c-fos expression in the nucleus accumbens that was significantly inhibited by TAT-D1 pretreatment. No effects of SKF 83959 were seen in caudate putamen. D1-D2 heteromer disruption by TAT-D1 did not have any effects in any striatal subregions, but induced significant c-fos immunoreactivity in a number of cortical regions including the orbitofrontal cortex, prelimbic and infralimbic cortices and piriform cortex. The induction of c-fos by TAT-D1 was also evident in the anterior olfactory nucleus, as well as the lateral habenula and thalamic nuclei. These findings show for the first time that the D1-D2 heteromer can differentially regulate c-fos expression in a region-dependent manner either through its activation or through tonic inhibition of neuronal activity.

3-D imaging, illustration, and quantitation of enteric glial network in transparent human colon mucosa.

BACKGROUND: Enteric glia form a network in the intestinal mucosa and have been suggested to engage in multidirectional interactions with the epithelium, blood vessels, nerves, and immune system. However, due to the dispersed nature of the glial network, standard histology cannot provide a global view of the network architecture. We prepared transparent human colon mucosa for three-dimensional (3-D) confocal microscopy with S100B immunostaining to reveal the location-dependent glial network for qualitative and quantitative analyses. METHODS: Full-thickness human colons were acquired from colectomies performed for colorectal cancer. We targeted the mucosa away from the tumor site to characterize the glial network morphology. Optical clearing (use of immersion solution to reduce scattering) was applied to generate transparent specimens for deep-tissue microscopy. KEY RESULTS: Two features of the glial network were seen: (i) A dense glial population resides at the crypt base/mucosal boundary in contact with the lymphatic vessels, and (ii) from the base, the glial network elongates along the crypt axis with peri-cryptic and peri-vascular connections toward the opening. We quantified the mucosal glia as the S100B-positive cells with at least two processes extending from the cell body. Examples of the global and in-depth imaging of adenoma were given to illustrate the morphological correlation between the loss of glial fibers and the aberrant crypts. CONCLUSIONS & INFERENCES: We have established a useful approach for 3-D imaging, panoramic illustration, and quantitation of the enteric glia in the human colon mucosa to help characterize their roles with mucosal components in health and disease.

Room temperature SnO2 thin film gas sensor fabricated on Si nanospikes.

The SnO2 thin film CO gas sensors have been fabricated on silicon nanostructured surface made using a femtosecond pulsed laser irradiation. The measurement shows a significant response to the CO gas at room temperature. While a SnO2 sensor fabricated on a flat surface shows no response when CO gas exists at room temperature. The high area/volume ratio and sharp structures of the nanospikes enhance the sensitivity of SnO2 at room temperature.

Inhibitory mechanisms of kinetin, a plant growth-promoting hormone, in platelet aggregation.

Kinetin has been shown to have anti-aging effects on several different systems including plants and human cells. The aim of this study was to examine the detailed inhibitory mechanisms of kinetin in platelet aggregation. In this study, kinetin concentration-dependently (50-150 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Kinetin (70 and 150 microM) also concentration-dependently inhibited intracellular Ca2+ mobilization and phosphoinositide breakdown in platelets stimulated by collagen (1 microg/ml). Kinetin (70 and 150 microM) significantly inhibited thromboxane A2 formation stimulated by collagen (1 microg/ml) and arachidonic acid (60 microM) in human platelets. In addition, kinetin (70 and 150 microM) significantly increased the formation of cyclic AMP. Intracellular pH values were measured spectrofluorometrically using the fluorescent probe BCECF-AM in platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of kinetin (70 and 150 microM). Rapid phosphorylation of a platelet protein of molecular weight (Mr) 47000 (P47), a marker of protein kinase C activation, was triggered by collagen (1 microg/ml). This phosphorylation was inhibited by kinetin (70 and 150 microM). In conclusion, these results indicate that the anti-platelet activity of kinetin may be involved in the following pathways: kinetin's effects may initially be due to inhibition of the activation of phospholipase C and the Na+/H+ exchanger. This leads to lower intracellular Ca2+ mobilization, followed by inhibition of TxA2 formation and then increased cyclic AMP formation, followed by a further inhibition of the Na+/H+ exchanger, ultimately resulting in markedly decreased intracellular Ca2+ mobilization and phosphorylation of P47. These results suggest that kinetin has an effective anti-platelet effect and that it may be a potential therapeutic agent for arterial thrombosis.

The hyperaggregability of platelets from normal pregnancy is mediated through thromboxane A2 and cyclic AMP pathways.

There is substantial evidence of increased platelet reactivity in vivo and in vitro during pregnancy, with the risk of developing pre-eclampsia. In this study, platelet function was studied during 28-40 weeks of gestation in a group of women who remained normotensive and in a group of nonpregnant female controls. Platelet aggregation stimulated by thrombin and adenosine diphosphate was markedly enhanced in washed platelets from pregnant subjects. Thrombin (0.04 U/ml)-evoked increases in intracellular Ca+2 mobilization of Fura 2-AM-loaded platelets were also enhanced in pregnant subjects. The binding of fluorescein isothiocyanate (FITC)-triflavin (2 microg/ml) to the glycoprotein IIb/IIIa complex in thrombin-activated platelets did not differ significantly between the nonpregnant and pregnant groups. Thromboxane A2 (TXA2) formation in both resting and thrombin-activated platelets from pregnant subjects was significantly greater than from nonpregnant subjects. Levels of cyclic adenosine monophosphate (cAMP) in both resting and prostaglandin E1-treated platelets (10 micromol/l) from pregnant subjects were significantly lower than those from nonpregnant subjects. There were no significant differences between nonpregnant and pregnant subjects in platelet cAMP levels in the presence of imidazole (600 micromol/l) and indomethacin (500 micromol/l). Intracellular pH values in platelets were measured spectrofluorometrically using the fluorescent probe, BCECF-AM. The increase in intracellular pH stimulated by thrombin (0.04 U/ml) in pregnant subjects was markedly greater than that in observed nonpregnant subjects. We conclude that the agonist-induced hyperaggregability of platelets in normal pregnancy may be due, at least partly, to stimulation of the Na+/H+ exchanger and subsequently to elevated intracellular Ca+2 mobilization, and then to increased TXA2 formation and a lowered level of cAMP, which leads to further increases in intracellular Ca+2 mobilization, and finally to enhanced platelet aggregation.

Vasoactive intestinal polypeptide and its receptor changes in human temporal lobe epilepsy.

The distribution of the VIP receptor in the human hippocampus was studied by receptor autoradiography using [3-iodotyrosyl-125I]Vasoactive Intestinal Peptide (VIP) as a ligand, and the relationship of receptor distribution to the distribution of the peptide (visualized by immunocytochemistry) was examined in hippocampi surgically removed from patients with medically intractable temporal lobe epilepsy (TLE) and hippocampi obtained at autopsy from neurologically normal subjects. In the autopsy hippocampi and hippocampi from TLE patients with extrahippocampal temporal lobe lesions [125I]VIP binding was highest in the dentate molecular layer, with lower levels in the fields of Ammon's Horn (CA fields) and the subiculum. In hippocampi from patients with no temporal lobe lesions but considerable hippocampal neuronal loss there were significant elevations in the levels of ligand binding in all CA fields and the subiculum. Ligand binding densities in all CA fields of the patient hippocampi were strongly negatively correlated with neuronal numbers. Immunocytochemical localization of VIP shows no obvious change in the distribution patters of VIP immunoreactivity in the patient groups. This is the first demonstration of VIP and its receptor distribution in the human hippocampus. It is suggested that the elevated levels of receptor binding in the hippocampal seizure focus may indicate a mechanism for greater excitability of neurons and/or for their survivability in the face of the increased excitation and potential for injury in a seizure focus.

T cells induce terminal differentiation of transformed B cells to mature plasma cell tumors.

Major interest in the analysis of mature plasma cell neoplasias of mice and humans has focused on identification of precursor cells that give rise to mature malignant plasma cells. Although several laboratories have recently suggested that such cells are present in the granulomas of pristane-treated mice and the bone marrow of some multiple myeloma patients, the in vivo cellular interactions required for their differentiation into mature plasma cell tumors remains unclear. Given the extensive interactions of peripheral T cells and normal B cells, we assessed the potential role of T cells in plasma-cell tumor development, by using a myc, raf-containing retrovirus, J3V1, to induce plasmacytomas in normal BALB/c mice, T-cell-deficient nude mice, and T-cell-reconstituted nude mice. The B-lineage tumors arising in normal BALB/c mice were uniformly mature plasmacytomas, most of which secreted immunoglobulin. In contrast, nude mice yielded predominantly non-immunoglobulin-secreting B-cell lymphomas with a phenotype characteristic of peripheral B cells. T-cell reconstitution of nude mice prior to tumor induction resulted in a shift from B-cell lymphomas to plasmacytomas. These results imply that transformation can occur prior to terminal differentiation of B cells and that such transformed cells can be driven to terminal differentiation by peripheral T cells. These findings further suggest that, in human multiple myeloma, the ability of T cells to influence the differentiation state of transformed B cells may provide a mechanism by which malignant plasma cells found in the bone marrow could arise from clonotypically related less-mature B cells found in both the bone marrow and periphery.

Immunophenotyping of 515 cases of acute lymphoblastic leukemia in China.

Using cell surface markers and a panel of monoclonal antibodies, 515 cases of acute lymphoblastic leukemia (ALL) were immunophenotyped. T cell type ALL (T-ALL), non-T cell type ALL (Non-T-ALL) including common ALL (C-ALL), Null-ALL and B cell type ALL (B-ALL) were found. These major subtypes of ALL were further divided according to their phenotypes in detail. It was noticed that the phenotypes of these subtypes of ALL reflected basically the phenotypes of normal T or B cells at various differentiation stages or certain population of lymphocytes. The diagnosis of cell lineage was more precise when based on immunophenotyping than morphological description. The combination of morphological and immunological classification can improve the diagnosis of acute leukemias. In addition, it was observed that the immunophenotyping was relevant to clinicopathologic features, responses to therapy and prognosis of ALL patients. The incidences of major subtypes of ALL, the age distribution of ALL subsets and male sex bias with T-ALL in Chinese are discussed.

[Correlation of immunophenotype with clinical features in T-cell acute lymphoblastic leukemia].

The immunophenotype of leukemicblasts from 111 patients with T-ALL or T-NHL were further examined by using a panel of standardized McAbs of CD nomenclature to human leukocyte differentiation antigens. Four major subsets of T-ALL were defined: pre T-ALL, immature T-ALL (I), common T-ALL (II) and mature T-ALL (III), with the percentages 20.7%, 20.7%, 20.7% and 37.0% respectively. In addition there was a case with M-T acute hybrid leukemia. Some of the clinical features of the patients with T-ALL and T-NHL were compared. It was found that male predominance, older age, higher leukocyte count, lower platelet level, relative higher hemoglobin level and increased incidence of extramedullary involvement, including hepatomegaly, splenomegaly and lymphadenopathy were alike for all subsets of T-ALL cases. However, the average white cell level and incidence of lymphadenopathy in the pre T-ALL subset significantly differed from those in other subsets. The correlation of immunophenotype with morphologic characterization was also discussed in this paper.

Hippocampal neuronal density in temporal lobe epilepsy with and without gliomas.

The majority of patients with temporal lobe epilepsy show hippocampal sclerosis, which pathologically represents neuronal loss and gliosis. We studied volumetric neuronal density on a representative mid to mid-posterior level slice of hippocampi surgically removed from intractable temporal lobe epilepsy cases, and compared the results between 25 non-tumor epilepsy (NTE) cases and 5 tumor-associated epilepsy (TAE) cases. Eleven age-matched non-epileptic autopsy cases were studied as controls. Cells were counted in the CA1 through CA4 fields and the stratum granulosum of the dentate fascia. In NTE every hippocampal field showed statistically significant loss of neurons, the neuronal density in each field ranging from 35% to 50% of that of control. The mean neuronal density between the TAE and NTE groups also showed statistically significant differences in all hippocampal fields. The neuronal density of hippocampal fields of NTE ranged from 43% to 58% of that of TAE. Tumor-associated epilepsy cases, however, failed to show any statistically significant deviation from the control in their neuronal density. The etiology of the difference in neuronal density between the TAE and NTE groups is discussed.