Machine learning-based MRI radiomics for assessing the level of tumor infiltrating lymphocytes in oral tongue squamous cell carcinoma: a pilot study.

BACKGROUND: To investigate the value of machine learning (ML)-based magnetic resonance imaging (MRI) radiomics in assessing tumor-infiltrating lymphocyte (TIL) levels in patients with oral tongue squamous cell carcinoma (OTSCC). METHODS: The study included 68 patients with pathologically diagnosed OTSCC (30 with high TILs and 38 with low TILs) who underwent pretreatment MRI. Based on the regions of interest encompassing the entire tumor, a total of 750 radiomics features were extracted from T2-weighted (T2WI) and contrast-enhanced T1-weighted (ceT1WI) imaging. To reduce dimensionality, reproducibility analysis by two radiologists and collinearity analysis were performed. The top six features were selected from each sequence alone, as well as their combination, using the minimum-redundancy maximum-relevance algorithm. Random forest, logistic regression, and support vector machine models were used to predict TIL levels in OTSCC, and 10-fold cross-validation was employed to assess the performance of the classifiers. RESULTS: Based on the features selected from each sequence alone, the ceT1WI models outperformed the T2WI models, with a maximum area under the curve (AUC) of 0.820 versus 0.754. When combining the two sequences, the optimal features consisted of one T2WI and five ceT1WI features, all of which exhibited significant differences between patients with low and high TILs (all P < 0.05). The logistic regression model constructed using these features demonstrated the best predictive performance, with an AUC of 0.846 and an accuracy of 80.9%. CONCLUSIONS: ML-based T2WI and ceT1WI radiomics can serve as valuable tools for determining the level of TILs in patients with OTSCC.

The genetic and immune features of salivary gland secretory carcinoma with high-grade transformation.

OBJECTIVES: To compare the clinicopathological, molecular, and immune features of conventional and high-grade transformation (HGT) secretory carcinoma (SC) in salivary glands. MATERIALS AND METHODS: The clinicopathological data of 88 cases including 74 conventional SCs and 14 SCs with HGT were reviewed. Targeted next-generation sequencing was performed in 11 SCs with HGT and 7 conventional SCs. The level of PD-L1 and CD8+ TILs was determined by immunohistochemistry. RESULTS: Compared with the conventional group, the rates of nodal metastasis, local recurrence, distant metastasis and mortality were significantly higher in the HGT cohort. Mutations of ARID1A/B, KMT2A, HOXD13, NRG1 and ETV6 genes were identified in HGT SCs. A recurrent E307G mutation in GATA6 gene was also observed in two cases. Two deceased HGT patients with distant metastasis harboured NOTCH3 mutations. ETV6-RET translocation was prone to occur in the HGT SCs. Additionally, PD-L1 expression was low, and CD8+ TILs were sparse in most HGT cases. CONCLUSION: Our findings reveal novel gene alterations involved in the progression of HGT in SCs. Most HGT SCs patients cannot benefit from PD-L1 blocking and may be approached with a distinct treatment strategy including the lymph node dissection and application of molecular target drugs in precision oncology.

A c-MET-Targeted Topical Fluorescent Probe cMBP-ICG Improves Oral Squamous Cell Carcinoma Detection in Humans.

INTRODUCTION: The postoperative survival of oral squamous cell carcinoma (SCC) relies on precise detection and complete resection of original tumors. The mucosal extension of the tumor is evaluated visually during surgery, but small and flat foci are difficult to detect. Real-time fluorescence imaging may improve detection of tumor margins. MATERIALS AND METHODS: In the current study, a peptide-based near-infrared (NIR) fluorescence dye, c-MET-binding peptide-indocyanine green (cMBP-ICG), which specifically targets tumor via c-MET binding, was synthetized. A prospective pilot clinical trial then was conducted with oral SCC patients and intraoperatively to assess the feasibility of cMBP-ICG used to detect tumors margins. Fluorescence was histologically correlated to determine sensitivity and specificity. RESULTS: The immunohistochemistry (IHC) results demonstrated increased c-Met expression in oral SCC compared with normal mucosa. Tumor-to-background ratios ranged from 2.71 ± 0.7 to 3.11 ± 1.2 in different concentration groups. From 10 patients with oral SCC, 60 specimens were collected from tumor margins. The sensitivity and specificity of discriminative value derived from cMBP-ICG application in humans were respectively 100% and 75%. CONCLUSIONS: Topical application of cMBP-ICG is feasible and safe for optimizing intraoperative visualization and tumor margin detection in oral SCC patients, which could clinically increase the probability of complete resections and improve oncologic outcomes.

Adenoid cystic carcinoma in children and young adults: A clinicopathological study of 12 cases.

OBJECTIVE: To investigate the clinicopathological characteristics, immunoprofile, and molecular alterations of adenoid cystic carcinoma (ACC) in children and young adults. MATERIALS AND METHODS: Twelve cases of ACC were included. MYB, MYBL1, Ki-67, type IV Collagen, Laminin, and LAMB1 expression were detected by immunohistochemistry. MYB and MYBL1 rearrangements were detected by fluorescence in situ hybridization. RESULTS: Among 12 patients, four were female and eight were male. Seven cases (58.3%) located in major salivary glands and eight cases (66.7%) were classified as Grade I. Ten tumors (83.3%) had collagenous and hyalinized stroma. MYB was positive in 83.3% cases, and the average Ki-67 labeling index (LI) was 8.3%. LAMB1, type IV Collagen, and Laminin were positive in 91.7%, 66.7%, and 58.3% cases, respectively. Besides, three out of eight tumors had MYB rearrangement. Cases without MYB rearrangement were negative for MYBL1 expression and MYBL1 rearrangement. The average follow-up time was 91.8 months. Four patients had recurrent diseases. CONCLUSIONS: ACC in children and young adults was seen more frequently in males and major salivary glands. Most cases had ECM and hyaline stroma. Grade III tumors, higher Ki-67 LI, negative expression of type IV Collagen, and Laminin showed a tendency of higher recurrence rate.

Atypical/unbalanced ETV6/NTRK3 rearrangement in salivary secretory carcinoma with a focus on the incidence, the patterns, and the clinical implications.

BACKGROUND: Atypical/unbalanced rearrangement in salivary secretory carcinoma was observed and its incidence, patterns, and clinical significance remain unknown. METHODS: One hundred and ninety-six cases of diagnosed secretory carcinoma were retrospectively reviewed. Fluorescence in situ hybridization for NTRK3/ETV6::NTRK3 was conducted on cases carrying the atypical ETV6 fluorescence in situ hybridization signals. Cases without ETV6::NTRK3 were selected for next-generation sequencing to reveal novel partner. Immunohistochemistry and follow-up were performed. RESULTS: Twenty-seven cases were confirmed to carry the atypical ETV6 fluorescence in situ hybridization signal patterns. The most common type of abnormality was the duplication of ETV6 5' end (1Y1GnR, n ≥ 2) with the incidence of 81.5% (22/27). Seventeen of 19 were identified with ETV6::NTRK3 and 2 with ETV6::RET. The immunophenotype was similar to the typical secretory carcinoma group. TrkC exhibited 68.8% sensitivity and 100% specificity for NTRK3 fusion. Microscopically, 5 out of 21 were accompanied by necrosis and 3 out of 21 showed neural invasion. Four out of 19 patients showed local relapse, 2 developed distant metastasis, and 1 died of disease. The patients with distant metastasis and even dead were both harbored ETV6::RET rearrangement. Statistical analysis revealed that there were no significant differences in disease-free survival, relapse-free survival, and distant metastasis-free survival between atypical and typical groups. CONCLUSION: Gene rearrangement can be identified although the fluorescence in situ hybridization signals were atypical, which was instructive for secretory carcinoma diagnosis in clinical practice. The signals of partners were also always atypical which may have an impact to the efficacy of targeted drugs. There was no statistical evidence that this group possessed worse prognosis. However, secretory carcinomas with ETV6::RET have dismal prognosis than those with ETV6::NTRK3.

Preclinical pharmacokinetics and metabolism of MAK683, a clinical stage selective oral embryonic ectoderm development (EED) inhibitor for cancer treatment.

MAK683 (N-((5-fluoro-2,3-dihydrobenzofuran-4-yl)methyl)-8-(2-methylpyridin-3-yl)-[1,2,4]triazolo[4,3-c]pyrimidin-5-amine) is a potent and orally bioavailable EED inhibitor for the potential treatment in oncology. Pharmacokinetics (PK) in preclinical species are characterised by low to moderate plasma clearances, high oral exposure, and moderate to high oral bioavailability at the dose of 1-2 mg/kg.A species comparison of the metabolic pathways of MAK683 has been made using [14C]MAK683 incubations with liver microsomes and hepatocytes from rat, dog, cynomolgus monkey, and human. Overall, the in vitro hepatic metabolism pathway of MAK683 in all five species was very complex. A total of 60 metabolites with 19 metabolites >1.5% of the total integrated area in the radiochromatogram of at least one species were identified in five species (rat, mouse, dog, monkey, and human).The primary in vitro hepatic oxidative metabolism pathway identified in humans involved 2-hydroxylation of the dihydrofuran ring to form alcohol (M28), which was in a chemical equilibrium favouring the formation of its aldehyde form. The aldehyde was then oxidised to the carboxylic acid metabolite (M26) or reduced to the O-hydroxyethylphenol (M29). N-dealkylation (M1), 3-hydroxylation of the dihydrofuran ring (M27), N-oxidation of the pyridine moiety (M53), and sulphate conjugation of M28 to form M19 were also important biotransformation pathways in human hepatocytes. The above major human hepatic metabolic pathways were also observed across the animal species (rat, mouse, dog, and monkey) mostly providing precursors for the formation of other metabolites via further oxygenation, glucuronidation, and sulphation pathways.No human-specific metabolites were observed. In addition, in vivo biotransformation was also conducted in bile-duct cannulated (BDC) rat. The metabolism in BDC rat was similar to those observed the in vitro hepatocytes.

Newly Emerged Antiviral Strategies for SARS-CoV-2: From Deciphering Viral Protein Structural Function to the Development of Vaccines, Antibodies, and Small Molecules.

Coronavirus disease 2019 (COVID-19) caused by the infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become the most severe health crisis, causing extraordinary economic disruption worldwide. SARS-CoV-2 is a single-stranded RNA-enveloped virus. The process of viral replication and particle packaging is finished in host cells. Viral proteins, including both structural and nonstructural proteins, play important roles in the viral life cycle, which also provides the targets of treatment. Therefore, a better understanding of the structural function of virus proteins is crucial to speed up the development of vaccines and therapeutic strategies. Currently, the structure and function of proteins encoded by the SARS-CoV-2 genome are reviewed by several studies. However, most of them are based on the analysis of SARS-CoV-1 particles, lacking a systematic review update for SARS-CoV-2. Here, we specifically focus on the structure and function of proteins encoded by SARS-CoV-2. Viral proteins that contribute to COVID-19 infection and disease pathogenesis are reviewed according to the most recent research findings. The structure-function correlation of viral proteins provides a fundamental rationale for vaccine development and targeted therapy. Then, current antiviral vaccines are updated, such as inactive viral vaccines and protein-based vaccines and DNA, mRNA, and circular RNA vaccines. A summary of other therapeutic options is also reviewed, including monoclonal antibodies such as a cross-neutralizer antibody, a constructed cobinding antibody, a dual functional monoclonal antibody, an antibody cocktail, and an engineered bispecific antibody, as well as peptide-based inhibitors, chemical compounds, and clustered regularly interspaced short palindromic repeats (CRISPR) exploration. Overall, viral proteins and their functions provide the basis for targeted therapy and vaccine development.

Expression profile of circular RNA and construction of circular RNA-Micro RNA network in salivary adenoid cystic carcinoma.

BACKGROUND: Circular RNAs (circRNAs) is a newly discovered type of non-coding RNA, the abnormal expression of which has been demonstrated in many types of human tumors. So they have been considered as promising candidates as diagnostic and therapeutic targets in cancer. This research aimed to screen the profile of circRNA expression in salivary adenoid cystic carcinoma (SACC). METHODS: Using the threshold of FDR < 0.05 and fold change > 2 or < 0.5, 5 up-regulated and 26 down-regulated circRNAs were identified. The reliability of sequencing was verified by the expression detection of randomly selected circRNAs via qRT-PCR. RESULTS: Moreover, the circRNA-miRNA system was established by bioinformatics approaches and successfully identified an interaction between circRNA ABCA13 and a cancer-related miRNA (miR-138-5p), which was also verified by qRT-PCR. Moreover, the predicted molecular interaction proved that circRNA ABCA13 may promote SACC through inhibition of miR-138-5p. CONCLUSIONS: Collectively, this study has offered the first report about the circRNA expression profile and circRNA-miRNA network in SACC. All of the above could benefit the exploration of novel therapeutic target in SACC treatment.

Clinicopathological aspects of primary mucosa-associated lymphoid tissue lymphoma of the salivary gland: A retrospective single-center analysis of 72 cases.

BACKGROUND: Salivary gland extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue lymphoma (MALT lymphoma) is uncommon and has not been studied extensively. We aimed to investigate the features of clinicopathological and molecular changes of salivary MALT lymphoma. METHODS: Seventy-two cases of primary salivary MALT lymphoma that had clinicopathological information available were utilized in this study. MALT1 gene translocation, trisomy 3, and trisomy 18 were detected by interphase fluorescence in situ hybridization (FISH). The data were analyzed using SPSS 17.0 software package. RESULTS: The ratio of male to female was 1:2.8, and the median age was 57.0 years. 12.5% (9/72) of the patients presented with multiple swellings. Among the others with solitary mass, the parotid gland was involved most frequently (47/63,74.6%), followed by the palate (7/63, 11.1%). 34.7% of patients had an autoimmune disease, with Sjögren syndrome (SS) being the most common. Among the 70 cases successfully performed, it was identified that trisomy 3 was the most frequent molecular change (41/70, 58.6%), followed by trisomy 18 (7/70, 10%) and MALT1 translocation (5/70, 7.1%). The tumor tissue tended to exhibit trisomy 3 in patients without SS (p = 0.038). The 5-year overall survival was 94.1%, and the 5-year disease-free survival was 85.3% (mean follow-up time: 104.7 months). The patients without SS and trisomy 18 had a prolonged recurrence-free survival (p = 0.015, p = 0.001 respectively). CONCLUSION: Salivary gland MALT lymphoma is associated with autoimmune diseases, and trisomy 3 is the most common genetic change. Trisomy 18 can be used to predict the possibility of tumor relapse.

Indicators for Malignant Transformation in Patients With Lymphoepithelial Lesion.

PURPOSE: Salivary lesion (LEL) represents a unique disease, and some patients have malignant transformations. The study aims were to estimate the frequency of malignant transformation and the subtype of the malignant component and to identify factors associated with malignant transformation and subtype of the malignant component in patients with LEL. PATIENTS AND METHODS: This study was based on a retrospective cohort study between 2005 and 2017 from patients who were diagnosed as LEL. The predictor variable was composed of a set of variables grouped into demographic, clinical, and pathologic features. The outcome variables were malignant transformation status and subtype of the malignant components. All parameters between the predictor variables and outcome variables were analyzed using the χ2 test and a logistic regression model. RESULTS: The sample was composed of 252 cases of LEL (including with or without malignant transformation) with a mean age of 50.3 years; 58 (58 of 252; 23.0%) were males, 194 (194 of 252; 77.0%) were females. The parotid gland was the most common site of LEL (206 of 252; 81.7%), and 36.5% (92 of 252) of the patients had a history of Sjögren syndrome (SS). Masses greater than 2 cm in diameter had evidence of malignant transformation (P < .001). Factors associated with the subtype of malignant components were a history of SS (P < .001) and Epstein-Barr virus infection. The percentages of nonmalignant transformations, LEL with extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT lymphoma), and LEL progressing to lymphoepithelial carcinoma were 44.8 (113 of 252), 47.6 (120 of 252), and 7.6% (19 of 252), respectively. CONCLUSIONS: More than half of cases have a malignant transformation, and MALT lymphoma is the most common malignant subtype. A larger mass (>2 cm) is an independent indicator of malignant transformation in LEL patients. History of SS among LEL patients is considered a risk factor for MALT lymphoma.

Current Options and Future Directions for NAFLD and NASH Treatment.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, with a broad spectrum ranging from simple steatosis to advanced stage of nonalcoholic steatohepatitis (NASH). Although there are many undergoing clinical trials for NAFLD treatment, there is no currently approved treatment. NAFLD accounts as a major causing factor for the development of hepatocellular carcinoma (HCC), and its incidence rises accompanying the prevalence of obesity and diabetes. Reprogramming of antidiabetic and anti-obesity medicine is a major treatment option for NAFLD and NASH. Liver inflammation and cellular death, with or without fibrosis account for the progression of NAFLD to NASH. Therefore, molecules and signaling pathways involved in hepatic inflammation, fibrosis, and cell death are critically important targets for the therapy of NAFLD and NASH. In addition, the avoidance of aberrant infiltration of inflammatory cytokines by treating with CCR antagonists also provides a therapeutic option. Currently, there is an increasing number of pre-clinical and clinical trials undergoing to evaluate the effects of antidiabetic and anti-obesity drugs, antibiotics, pan-caspase inhibitors, CCR2/5 antagonists, and others on NAFLD, NASH, and liver fibrosis. Non-invasive serum diagnostic markers are developed for fulfilling the need of diagnostic testing in a large amount of NAFLD cases. Overall, a better understanding of the underlying mechanism of the pathogenesis of NAFLD is helpful to choose an optimized treatment.

Ameloblastoma with mucous cells: A clinicopathological, BRAF mutation, and MAML2 rearrangement study.

OBJECTIVES: To investigate the clinicopathological features, BRAF V600E mutation, and MAML2 rearrangement of ameloblastoma with mucous cell differentiation. MATERIALS AND METHODS: Five cases of ameloblastoma with mucous cell differentiation were retrospectively studied. Clinicopathological features, BRAF V600E mutation, and MAML2 rearrangement were analyzed. Follow-up information was available for all cases. RESULTS: Of five cases, two cases were male and three were female, aged 18-55 years. Four cases were located in the mandible and one case in the maxilla. Histologically, four of the five cases (80%) presented with cystic features and three of the five cases (60%) with varying degrees of squamous metaplasia. The mucous cells were located in the epithelial islands or the luminal aspect of the cystic cavities. The BRAF V600E mutation was found in three of five cases (60%). All the cases showed no MAML2 rearrangement. Two cases were recurrent lesions, and one case had a local recurrence during the follow-up. CONCLUSIONS: Ameloblastoma with mucous cell differentiation is closely related to the cystic features, squamous metaplasia, and shows a high prevalence of BRAF V600E mutation. The absence of MAML2 rearrangement reveals that ameloblastoma with mucous cell differentiation and central mucoepidermoid carcinoma (MEC) are two distinct tumor entities.

Clinicopathologic Features and Prognostic Factors of Widely Invasive Carcinoma Ex Pleomorphic Adenoma of Parotid Gland: A Clinicopathologic Analysis of 126 Cases in a Chinese Population.

PURPOSE: Salivary intracapsular carcinoma ex pleomorphic adenoma (ICCXPA) and minimally invasive CXPA (MICXPA) generally have favorable outcomes. In contrast, widely invasive CXPA (WICXPA) frequently results in disease-related death. The aims of the present study were to analyze the differences in the clinicopathologic features between parotid gland ICCXPA or MICXPA and WICXPA and the prognostic factors for WICXPA. MATERIALS AND METHODS: We performed a retrospective cohort study. The clinicopathologic parameters of patients with primary CXPA of the parotid gland from our 2001 to 2012 cohort were reviewed. The predictor variable was a set of heterogeneous factors grouped into demographic, clinical, and pathologic features. The primary outcome variable was the tumor diagnosis, grouped into 3 categories: ICCXPA, MICXPA, and WICXPA. For statistical analysis, ICCXPA and MICXPA were combined into 1 group, with WICXPA analyzed separately. The differences in the clinicopathologic parameters between the 2 groups (ICCXPA plus MICXPA vs WICXPA) were evaluated using the χ2 test or t test. The secondary outcome variable was disease-specific survival (DSS) of those with WICXPA. The survival data for WICXPA were statistically analyzed using the Kaplan-Meier method and Cox regression. RESULTS: A total of 241 cases of CXPA had been diagnosed, including 63 cases of ICCXPA, 52 cases of MICXPA, and 126 cases of WICXPA. The patients with WICXPA were older than were those with ICCXPA/MICXPA (59.6 vs 51.4 years; P < .001) and had a larger tumor diameter (3.9 vs 3.3 cm; P = .040). The proportion of histologic high-grade tumor (P < .001), proportion of carcinoma more than 50% (P < .001), and proportion of lymph node involvement (P < .001) was greater in those with WICXPA. Cox regression analysis indicated that age, T stage, and N stage were independent prognostic factors of DSS for those with WICXPA. CONCLUSIONS: Older age, later T stage, a greater proportion of carcinoma, histologic high-grade findings, and lymph node involvement were associated with parotid gland WICXPA. Age, T stage, and N stage were the important independent factors for predicting the prognosis of patients with parotid gland WICXPA.

Application of fungal fluorescent staining in oral candidiasis: diagnostic analysis of 228 specimens.

BACKGROUND: Several conventional methods, including fungal culture and periodic acid-Schiff (PAS) reagent staining, have been used to diagnose oral candidiasis. The aim of this study was to evaluate the efficacy of a novel method, fungal fluorescent staining, in relation to conventional protocols in the diagnosis of oral candidiasis. METHODS: We collected 106 oral swabs and 122 oral biopsy tissues from patients highly suspected with oral candidiasis. We applied fungal culture and periodic acid-Schiff reagent staining as the gold standard diagnostic tools. The efficacy of these methods in determining the presence of Candida was compared with that of fluorescent staining. RESULTS: In the majority of specimens subjected to fluorescent staining, fungal organisms were distinguished by blue fluorescence surrounding their tubular or annular shapes. The sensitivity, specificity, Youden index, positive predictive value and negative predictive value of the fluorescent staining method were 82.7, 93.5, 76.7, 96.8 and 69.1% in oral swabs and 90.0, 92.9, 82.9, 96.0 and 82.9% in oral biopsy tissues, respectively. CONCLUSIONS: Fungal fluorescent staining represents a rapid method for detection of Candida, supporting its potential utility as an effective early diagnostic tool for oral candidiasis.

Antimicrobial efficacy and toxicity of novel CAMPs against P. aeruginosa infection in a murine skin wound infection model.

BACKGROUND: Treatment of P. aeruginosa wound infection is challenging due to its inherent and acquired resistance to many conventional antibiotics. Cationic antimicrobial peptides (CAMPs) with distinct modes of antimicrobial action have been considered as the next-generation therapeutic agents. In the present study, a murine skin surgical wound infection model was used to evaluate the in vivo toxicity and efficacy of two newly designed antimicrobial peptides (CAMP-A and CAMP-B), as chemotherapeutic agents to combat P. aeruginosa infection. RESULTS: In the first trial, topical application of CAMPs on the wounds at a dose equivalent to 4 × MIC for 7 consecutive days did not cause any significant changes in the physical activities, hematologic and plasma biochemical parameters, or histology of systemic organs of the treated mice. Daily treatment of infected wounds with CAMP-A and CAMP-B for 5 days at a dose equivalent to 2× MIC resulted in a significant reduction in wound bacterial burden (CAMP-A: 4.3 log10CFU/g of tissue and CAMP-B: 5.8 log10CFU/g of tissue), compared to that of the mock-treated group (8.1 log10CFU/g of tissue). Treatment with CAMPs significantly promoted wound closure and induced epidermal cell proliferation. Topical application of CAMP-A on wounds completely prevented systemic dissemination of P. aeruginosa while CAMP-B blocked systemic infection in 67% of mice and delayed the onset of systemic infection by at least 2 days in the rest of the mice (33%). In a second trial, daily application of CAMP-A at higher doses (5× MIC and 50× MIC) didn't show any significant toxic effect on mice and the treatments with CAMP-A further reduced wound bacterial burden (5× MIC: 4.5 log10CFU/g of tissue and 50× MIC: 3.8 log10CFU/g of tissue). CONCLUSIONS: The data collectively indicated that CAMPs significantly reduced wound bacterial load, promoted wound healing, and prevented hepatic dissemination. CAMP-A is a promising alternative to commonly used antibiotics to treat P. aeruginosa skin infection.

Can pattern and depth of invasion predict lymph node relapse and prognosis in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a special type of oral cancer. Cervical lymph node relapse may occur in a large percentage of TSCC patients, which usually indicates poor prognosis. In this cohort study, we focused on the predictive value of the pathological features on cervical lymph node relapse and TSCC prognosis (disease free survival). METHODS: One hundred forty-one TSCC patients staged as T1-2N0 were enrolled and categorized. Subjects were followed-up for 60 months. Univariate analysis was performed with Chi-square test for cervical lymph node relapse and Kaplan-Meier survival analysis and log rank P value for patient prognosis; multivariate analysis was also utilized with Cox regression. RESULTS: In univariate analysis, trabes growth pattern, depth of invasion greater than 4 mm, poor pathological differentiation and neurovascular invasion were considered as risk factors for cervical lymph node relapse and poor prognosis. In multivariate analysis, only patients with trabes growth pattern in the invasive front or depth of invasion larger than 4 mm had a higher risk of metastasis. Elder age group and trabes growth pattern of invasive front were considered as predictors of poor prognosis. Bad habits of smoking and alcohol consumption were related to the higher risk of metastasis. CONCLUSION: Trabes growth pattern of invasive front was a potent risk factor for TSCC cervical lymph node relapse and indicated poor prognosis. Preventive therapy including selective neck dissection was thus suggested for certain patients. TRIAL REGISTRATION: Not applicable.

Beta-defensin derived cationic antimicrobial peptides with potent killing activity against gram negative and gram positive bacteria.

BACKGROUND: Avian ß-defensins (AvBD) are cationic antimicrobial peptides (CAMP) with broad-spectrum antimicrobial activity, chemotactic property, and low host cytotoxicity. However, their bactericidal activity is greatly compromised under physiological salt concentrations which limits the use of these peptides as therapeutic agents. The length and the complex structure involving three conserved disulfide bridges are additional drawbacks associated with high production cost. In the present study, short linear CAMPs (11 to 25 a.a. residues) were developed based on the key functional components of AvBDs with additional modifications. Their biological functions were characterized. RESULTS: CAMP-t1 contained the CCR2 binding domain (N-terminal loop and adjacent α-helix) of AvBD-12 whereas CAMP-t2 comprised the key a.a. residues responsible for the concentrated positive surface charge and hydrophobicity of AvBD-6. Both CAMP-t1 and CAMP-t2 demonstrated strong antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus pseudintermedius. However, CAMP-t1 failed to show chemotactic activity and CAMP-t2, although superior in killing Staphylococcus spp., remained sensitive to salts. Using an integrated design approach, CAMP-t2 was further modified to yield CAMP-A and CAMP-B which possessed the following characteristics: α-helical structure with positively and negatively charged residues aligned on the opposite side of the helix, lack of protease cutting sites, C-terminal poly-Trp tail, N-terminal acetylation, and C-terminal amidation. Both CAMP-A and CAMP-B demonstrated strong antimicrobial activity against multidrug-resistant P. aeruginosa and methicillin-resistant S. pseudintermedius (MRSP) strains. These peptides were resistant to major proteases and fully active at physiological concentrations of NaCl and CaCl2. The peptides were minimally cytotoxic to avian and murine cells and their therapeutic index was moderate (≥ 4.5). CONCLUSIONS: An integrated design approach can be used to develop short and potent antimicrobial peptides, such as CAMP-A and CAMP-B. The advantageous characteristics, including structural simplicity, resistance to salts and proteases, potent antimicrobial activity, rapid membrane attacking mode, and moderate therapeutic index, suggest that CAMP-A and CAMP-B are excellent candidates for development as therapeutic agents against multidrug-resistant P. aeruginosa and methicillin-resistant staphylococci.

Novel synthetic analogues of avian ß-defensin-12: the role of charge, hydrophobicity, and disulfide bridges in biological functions.

BACKGROUND: Avian ß-defensins (AvBD) possess broad-spectrum antimicrobial, LPS neutralizing and chemotactic properties. AvBD-12 is a chemoattractant for avian immune cells and mammalian dendritic cells (JAWSII) - a unique feature that is relevant to the applications of AvBDs as chemotherapeutic agents in mammalian hosts. To identify the structural components essential to various biological functions, we have designed and evaluated seven AvBD analogues. RESULTS: In the first group of analogues, the three conserved disulfide bridges were eliminated by replacing cysteines with alanine and serine residues, peptide hydrophobicity and charge were increased by changing negatively charged amino acid residues to hydrophobic (AvBD-12A1) or positively charged residues (AvBD-12A2 and AvBD-12A3). All three analogues in this group showed improved antimicrobial activity, though AvBD-12A3, with a net positive charge of +9, hydrophobicity of 40% and a predicted CCR2 binding domain, was the most potent antimicrobial peptide. AvBD-12A3 also retained more than 50% of wild type chemotactic activity. In the second group of analogues (AvBD-12A4 to AvBD-12A6), one to three disulfide bridges were removed via substitution of cysteines with isosteric amino acids. Their antimicrobial activity was compromised and chemotactic activity abolished. The third type of analogue was a hybrid that had the backbone of AvBD-12 and positively charged amino acid residues AvBD-6. The antimicrobial and chemotactic activities of the hybrid resembled that of AvBD-6 and AvBD-12, respectively. CONCLUSIONS: While the net positive charge and charge distribution have a dominating effect on the antimicrobial potency of AvBDs, the three conserved disulfide bridges are essential to the chemotactic property and the maximum antimicrobial activity. Analogue AvBD-12A3 with a high net positive charge, a moderate degree of hydrophobicity and a CCR2-binding domain can serve as a template for the design of novel antimicrobial peptides with chemotactic property and salt resistance.

Methylcrotonyl-CoA Carboxylase Regulates Triacylglycerol Accumulation in the Model Diatom Phaeodactylum tricornutum.

The model marine diatom Phaeodactylum tricornutum can accumulate high levels of triacylglycerols (TAGs) under nitrogen depletion and has attracted increasing attention as a potential system for biofuel production. However, the molecular mechanisms involved in TAG accumulation in diatoms are largely unknown. Here, we employed a label-free quantitative proteomics approach to estimate differences in protein abundance before and after TAG accumulation. We identified a total of 1193 proteins, 258 of which were significantly altered during TAG accumulation. Data analysis revealed major changes in proteins involved in branched-chain amino acid (BCAA) catabolic processes, glycolysis, and lipid metabolic processes. Subsequent quantitative RT-PCR and protein gel blot analysis confirmed that four genes associated with BCAA degradation were significantly upregulated at both the mRNA and protein levels during TAG accumulation. The most significantly upregulated gene, encoding the ß-subunit of methylcrotonyl-CoA carboxylase (MCC2), was selected for further functional studies. Inhibition of MCC2 expression by RNA interference disturbed the flux of carbon (mainly in the form of leucine) toward BCAA degradation, resulting in decreased TAG accumulation. MCC2 inhibition also gave rise to incomplete utilization of nitrogen, thus lowering biomass during the stationary growth phase. These findings help elucidate the molecular and metabolic mechanisms leading to increased lipid production in diatoms.