Influence of PD-1/PD-L1 on immune microenvironment in oral leukoplakia and oral squamous cell carcinoma.

OBJECTIVE: To evaluate the relation between the expression of PD-1, PD-L1, CD3, CD8, Foxp3 and clinicopathological features in patients with oral leukoplakia (OLK) and oral squamous cell carcinomas (OSCC) as well as the malignant outcome in OLK patients, and to study the effect of PD-1 and PD-L1 on immune microenvironment in the progression of oral carcinogenesis. METHODS: We evaluated the expression of PD-1/PD-L1 and composition of CD3+ , CD8+ and Foxp3+ T lymphocytes in OLK and OSCC samples by immunohistochemical (IHC) staining and analyzed their relation with clinical information and malignant transformation in OLK patients. RESULTS: IHC staining demonstrated that the expression of PD-1 was significantly increased in the high-grade OLK group than in the low-grade OLK group, while PD-L1 was detected mainly in OSCC. The expression of CD3, CD8, and Foxp3 was found higher in the high-grade OLK group than in the low-grade OLK group, and the Foxp3+ cells were found more in the OSCC group than in the high-grade OLK group. PD-1 was significantly correlated with CD3 (p < 0.05, R = 0.52), CD8 (p < 0.05, R = 0.46), and Foxp3 (p < 0.05, R = 0.46), and the low PD-1-expression group showed a better malignant-free survival than high PD-1 expression group in the OLK (p < 0.05). CONCLUSION: The PD-1/PD-L1 may induce immune suppression in OLK and accelerate the progress of malignant transformation.

Biallelic mutations in PMFBP1 cause acephalic spermatozoa.

The majority of men with defects in spermatogenesis remain undiagnosed. Acephalic spermatozoa is one of the diseases causing primary infertility. However, the causes underlying over half of affected cases remain unclear. Here, we report by whole-exome sequencing the identification of homozygous and compound heterozygous truncating mutations in PMFBP1 of two unrelated individuals with acephalic spermatozoa. PMFBP1 was highly and specifically expressed in human and mouse testis. Furthermore, immunofluorescence staining in sperm from a normal control showed that PMFBP1 localizes to the head-flagella junction region, and the absence of PMFBP1 was confirmed in patients harboring PMFBP1 mutations. In addition, we generated Pmfbp1 knock-out (KO) mice, which we found recapitulate the acephalic sperm phenotype. Label-free quantitative proteomic analysis of testicular sperm from Pmfbp1 KO and control mice showed 124 and 35 proteins, respectively, increased or decreased in sperm from KO mice compared to that found in control mice. Gene ontology analysis indicates that the biological process of Golgi vesicle transport was the most highly enriched in differentially expressed proteins, indicating process defects related to Golgi complex function may disturb formation of the head-neck junction. Collectively, our data indicate that PMFBP1 is necessary for sperm morphology in both humans and mice, and that biallelic truncating mutations in PMFBP1 cause acephalic spermatozoa.

Single Particle Tracking: From Theory to Biophysical Applications.

After three decades of developments, single particle tracking (SPT) has become a powerful tool to interrogate dynamics in a range of materials including live cells and novel catalytic supports because of its ability to reveal dynamics in the structure-function relationships underlying the heterogeneous nature of such systems. In this review, we summarize the algorithms behind, and practical applications of, SPT. We first cover the theoretical background including particle identification, localization, and trajectory reconstruction. General instrumentation and recent developments to achieve two- and three-dimensional subdiffraction localization and SPT are discussed. We then highlight some applications of SPT to study various biological and synthetic materials systems. Finally, we provide our perspective regarding several directions for future advancements in the theory and application of SPT.

A novel mutation in the STK11 gene causes heritable Peutz-Jeghers syndrome - a case report.

BACKGROUND: Peutz-Jeghers syndrome (PJS) is a rare disorder characterized by multiple gastrointestinal hamartomatous polyps and mucocutaneous pigmentation. STK11 has been identified as a causative gene for this disease. CASE PRESENTATION: Herein we report a Chinese Han kindred with PJS. Onset for the PJS signs in three of the patients was rarely as early as at birth. We identified a novel heterozygous mutation (c.440_441delGT, p.Arg147Leufs*15) in the gene STK11, causing a short frameshift followed by a deletion of 63% of the amino acids in the STK protein. This mutation co-segregated with the PJS phenotype, and was absent in two hundred of unrelated ethnicity-matched controls. The mutation led to expression decrease of unaffected STK11 protein in patients than in controls, as well in PJ polyps than in circulating leucocytes from the patients. Phosphorylation levels of the downstream kinase AMPKα altered according with the expression of STK11. These results indicated the possibility that haploinsufficiency and epigenetic reduction of STK11 contributed to the pathogenesis of the disease. CONCLUSION: This study identifies a novel mutation in the pathogenic gene STK11 leading to PJS.

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations.

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Single-Molecule Kinetics of Protein Adsorption on Thin Nylon-6,6 Films.

Understanding and controlling protein adsorption on surfaces is critical to a range of biological and materials applications. Kinetic details that provide the equilibrium and nonequilibrium mechanisms are difficult to acquire. In this work, single-molecule fluorescence microscopy was used to study the adsorption of Alexa 555 labeled α-lactalbumin (α-LA) on two chemically identical but morphologically different polymer surfaces: flat and porous nylon-6,6 thin films. The adsorption kinetics of spatially resolved single molecule α-LA binding to nylon films were quantified by a monolayer adsorption model. The surface morphology of the porous nylon-6,6 films increased the number of adsorption sites but decreased the binding affinity compared to the flat films. Such single-molecule based kinetic studies may be extended to various protein-polymer interactions.

Investigation of associations between ten polymorphisms and the risk of coronary artery disease in Southern Han Chinese.

A large-scale meta-analysis of 14 genome-wide association studies has identified and replicated a series of susceptibility polymorphisms for coronary artery disease (CAD) in European ancestry populations, but evidences for the associations of these loci with CAD in other ethnicities remain lacking. Herein we investigated the associations between ten (rs579459, rs12413409, rs964184, rs4773144, rs2895811, rs3825807, rs216172, rs12936587, rs46522 and rs3798220) of these loci and CAD in Southern Han Chinese (CHS). Genotyping was performed in 1716 CAD patients and 1572 controls using mass spectrography. Both allelic and genotypic associations of rs964184, rs2895811 and rs3798220 with CAD were significant, regardless of adjustment for covariates of gender, age, hypertension, type 2 diabetes, blood lipid profiles and smoking. Significant association of rs12413409 was initially not observed, but after the adjustment for the covariates, both allelic and genotypic associations were identified as significant. Neither allelic nor genotypic association of the other six polymorphisms with CAD was significant regardless of the adjustment. Our results indicated that four loci of the total 10 were associated with CAD in CHS. Therefore, some of the CAD-related loci in European ancestry populations are indeed susceptibility loci for the risk of CAD in Han Chinese.

Conformational transitions in the glycine-bound GluN1 NMDA receptor LBD via single-molecule FRET.

The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called "step transition and state identification" to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question.

Excitonic energy migration in conjugated polymers: the critical role of interchain morphology.

Excitonic energy migration was studied using single molecule spectroscopy of individual conjugated polymer (CP) chains and aggregates. To probe the effect of interchain morphology on energy migration in CP, tailored interchain morphologies were achieved using solvent vapor annealing to construct polymer aggregates, which were then studied with single aggregate spectroscopy. We report that highly ordered interchain packing in regioregular poly(3-hexylthiophene) (rr-P3HT) enables long-range interchain energy migration, while disordered packing in regiorandom poly(3-hexylthiophene) (rra-P3HT), even in aggregates of just a few chains, can dramatically impede the interchain mechanism. In contrast to rr-P3HT, interchain energy migration in poly(3-(2'-methoxy-5'-octylphenyl)thiophene) (POMeOPT), a polythiophene derivative with bulky side chains, can be completely inhibited. We use simulated structures to show that the reduction in interchain coupling is not due simply to increased packing distance between backbones of different chains, but reflects inhibition of stacking due to side-chain-induced twisting of the contours of individual chains. A competition from intrachain coupling has also been demonstrated by comparing POMeOPT aggregates with different polymer chain sizes.

Charge-dependent transport switching of single molecular ions in a weak polyelectrolyte multilayer.

The tunable nature of weak polyelectrolyte multilayers makes them ideal candidates for drug loading and delivery, water filtration, and separations, yet the lateral transport of charged molecules in these systems remains largely unexplored at the single molecule level. We report the direct measurement of the charge-dependent, pH-tunable, multimodal interaction of single charged molecules with a weak polyelectrolyte multilayer thin film, a 10 bilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) PAA/PAH. Using fluorescence microscopy and single-molecule tracking, two modes of interaction were detected: (1) adsorption, characterized by the molecule remaining immobilized in a subresolution region and (2) diffusion trajectories characteristic of hopping (D ∼ 10(-9) cm(2)/s). Radius of gyration evolution analysis and comparison with simulated trajectories confirmed the coexistence of the two transport modes in the same single molecule trajectories. A mechanistic explanation for the probe and condition mediated dynamics is proposed based on a combination of electrostatics and a reversible, pH-induced alteration of the nanoscopic structure of the film. Our results are in good agreement with ensemble studies conducted on similar films, confirm a previously-unobserved hopping mechanism for charged molecules in polyelectrolyte multilayers, and demonstrate that single molecule spectroscopy can offer mechanistic insight into the role of electrostatics and nanoscale tunability of transport in weak polyelectrolyte multilayers.

Troika of single particle tracking programing: SNR enhancement, particle identification, and mapping.

Single particle tracking (SPT) techniques provide a microscopic approach to probe in vivo and in vitro structure and reactions. Automatic analysis of SPT data with high efficiency and accuracy spurs the development of SPT algorithms. In this perspective, we review a range of available techniques used in SPT analysis programs. In addition, we present an example SPT program step-by-step to provide a guide so that researchers can use, modify, and/or write a SPT program for their own purposes.

[Death caused by anaphylactic shock: a forensic pathological analysis of 142 cases].

OBJECTIVE: To explore the forensic pathological features of death caused by anaphylactic shock. METHODS: One hundred and forty-two death cases of anaphylactic shock were retrospectively analyzed. The IgE level in the serum of anaphylactic shock cases were statistically compared with that of 62 non-anaphylactic shock cases. RESULTS: Most cases (77.46%) of anaphylactic shock death occurred in the medical institutes, with intravenous drug administration accounting for 53.53% of anaphylactic shock death. ß-Lactam antibiotics, glucocorticoid and herbal medications were responsible for a significant proportion of such cases. Although characteristic histopathological changes were absent in vast majority of these anaphylactic shock cases, the differences of IgE levels in the serum between anaphylactic shock group and non-anaphylactic shock group were statistically significant (P<0.05). CONCLUSION: Combined information including clinical data, autopsy results, IgE level, and other specific test results should be evaluated together in the forensic pathological diagnosis of anaphylactic shock.

Improved analysis for determining diffusion coefficients from short, single-molecule trajectories with photoblinking.

Two maximum likelihood estimation (MLE) methods were developed for optimizing the analysis of single-molecule trajectories that include phenomena such as experimental noise, photoblinking, photobleaching, and translation or rotation out of the collection plane. In particular, short, single-molecule trajectories with photoblinking were studied, and our method was compared to existing analytical techniques applied to simulated data. The optimal method for various experimental cases was established, and the optimized MLE method was applied to a real experimental system: single-molecule diffusion of fluorescent molecular machines known as nanocars.

[Sudden death caused by aortic dissection: 63 cases of forensic pathological analysis].

OBJECTIVE: To explore the cause of death, clinical manifestations and forensic pathological features of death cases caused by aortic dissection. METHODS: Sixty-three cases of aortic dissection were selected from forensic medical center, Sun Yat-sen University from 2001 to 2011 and retrospectively analyzed. RESULTS: The patients were mostly young and middle-aged male, aged from 30 to 49 years old. The DeBakey type II was the most common pathological type and the main cause of death was pericardial tamponade. The most common symptom was abdominal pain. However, the location of aorta dissection did not always correlate with the location of pain. Some cases showed no obvious clinical symptoms. The rupture was usually located in ascending aorta with atherosclerosis and pathological changes of hypertension. CONCLUSION: It is significant for diagnosis and evaluation the cause of death of aortic dissection by knowing the clinical symptoms and forensic pathological features.

Possible inhibitory molecular mechanism of farnesol on the development of fluconazole resistance in Candida albicans biofilm.

Candida albicans biofilm infections are usually treated with azole antifungals such as fluconazole. However, the development of resistance to this drug in C. albicans biofilms is very common, especially in immunocompromised individuals. The upregulation of the sterol biosynthetic pathway gene ERG and the efflux pump genes CDR and MDR may contribute to this azole tolerance in Candida species. We hypothesize that farnesol, an endogenous quorum sensing molecule with possible antimicrobial properties which is also the precursor of ergosterols in C. albicans, may interfere with the development of fluconazole resistance in C. albicans biofilms. To test this hypothesis, MICs were compared and morphology changes were observed by confocal laser scanning microscopy (CLSM) for farnesol-treated and -untreated and fluconazole-resistant groups. The expression of possible target genes (ERG11, ERG25, ERG6, ERG5, ERG3, ERG1, MDR1, CDR1, and CDR2) in biofilms was analyzed by reverse transcription-PCR (RT-PCR) and quantitative PCR (qPCR) to investigate the molecular mechanisms of the inhibitory effects of farnesol. The results showed a decreased MIC of fluconazole and thinner biofilms for the farnesol-treated group, indicating that farnesol inhibited the development of fluconazole resistance. The sterol biosynthetic pathway may contribute to the inhibitory effects of farnesol, as the transcription levels of the ERG11, ERG25, ERG6, ERG3, and ERG1 genes decreased in the farnesol-treated group.

[KCNQ1, KCNH2, KCNE1 and KCNE2 potassium channels gene variants in sudden manhood death syndrome].

OBJECTIVE: To investigate KCNQ1, KCNH2, KCNE1 and KCNE2 gene variants in the cases of sudden manhood death syndrome (SMDS). METHODS: One hundred and sixteen sporadic cases of SMDS and one hundred and twenty-five healthy controlled samples were enrolled. Genomic DNA was extracted from blood samples. Gene variants of KCNQ1, KCNH2, KCNE1 and KCNE2 were screened by direct sequencing. RESULTS: A total of 14 mutations and 14 SNP were detected. Two non-synonymous mutations of them were newfound. There was no non-synonymous mutation found in the control group. CONCLUSION: There are KCNQ1, KCNH2, KCNE1 and KCNE2 gene variants found in Chinese SMDS cases. KCNQ1, KCNH2, KCNE1 and KCNE2 gene mutation may correlate partly with the occurrence of some cases of the SMDS in China.

Psychological Stress and Functional Endometrial Disorders: Update of Mechanism Insights.

The human endometrium plays a vital role in providing the site for embryo implantation and maintaining the normal development and survival of the embryo. Recent studies have shown that stress is a common factor for the development of unexplained reproductive disorders. The nonreceptive endometrium and disturbed early maternal-fetal interaction might lead to infertility including the repeated embryo implantation failure and recurrent spontaneous abortion, or late pregnancy complications, thereby affecting the quality of life as well as the psychological status of the affected individuals. Additionally, psychological stress might also adversely affect female reproductive health. In recent years, several basic and clinical studies have tried to investigate the harm caused by psychological stress to reproductive health, however, the mechanism is still unclear. Here, we review the relationship between psychological stress and endometrial dysfunction, and its consequent effects on female infertility to provide new insights for clinical therapeutic interventions in the future.

The role of tyrosine phosphatase Shp2 in spermatogonial differentiation and spermatocyte meiosis.

The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.

The feasibility of measuring renal blood flow using transesophageal echocardiography in patients undergoing cardiac surgery.

BACKGROUND: There is no reliable method to monitor renal blood flow intraoperatively. In this study, we evaluated the feasibility and reproducibility of left renal blood flow measurements using transesophageal echocardiography during cardiac surgery. METHODS: In this prospective noninterventional study, left renal blood flow was measured with transesophageal echocardiography during three time points (pre-, intra-, and postcardiopulmonary bypass) in 60 patients undergoing cardiac surgery. Sonograms from 6 subjects were interpreted by 2 blinded independent assessors at the time of acquisition and 6 mo later. Interobserver and intraobserver reproducibility were quantified by calculating variability and intraclass correlation coefficients. RESULTS: Patients with Doppler angles of >30 degrees (20 of 60 subjects) were eliminated from renal blood flow measurements. Left renal blood flow was successfully measured and analyzed in 36 of 60 (60%) subjects. Both interobserver and intraobserver variability were <10%. Interobserver and intraobserver reproducibility in left renal blood flow measurements were good to excellent (intraclass correlation coefficients 0.604-0.999). Left renal arterial luminal diameter for the pre, intra, and postcardiopulmonary bypass phases, ranged from 3.8 to 4.1 mm, renal arterial velocity from 25 to 35 cm/s, and left renal blood flow from 192 to 299 mL/min. CONCLUSION: In patients undergoing cardiac surgery, it was feasible in 60% of the subjects to measure left renal blood flow using intraoperative transesophageal echocardiography. The interobserver and intraobserver reproducibility of renal blood flow measurements was good to excellent.

TLR2 deficiency enhances susceptibility to oral carcinogenesis by promoting an inflammatory environment.

Inflammation is closely related to oral squamous cell carcinoma (OSCC). However, its mechanism is still obscure. Toll-like receptor 2 (TLR2) plays an important role in oral chronic inflammatory diseases, but the role of TLR2 in OSCC is unclear. Here, we investigated the expression of TLR2 expression in OSCCs and examined the potential role of TLR2 in OSCC through its association with clinicopathological features and patient outcome. We used 4-nitroquinoline 1-oxide (4-NQO) to induce a tongue cancer model in TLR2-/- and wild type (WT) mice. Histological and clinical results both indicated that TLR2 played a protective role in oral tumorigenesis. The results of a cytometric bead array (CBA) indicated that TLR2 deficiency resulted in Th1 and Th2 cytokine abnormalities, especially Th2 abnormalities. Immunohistochemistry also showed that TLR2 deficiency increases the number of tongue-infiltrating M2 macrophages. Overall, our results demonstrated that TLR2 plays an important role in the prevention of oral tumorigenesis and affects the levels of Th2 cytokines and tongue-infiltrating M2 macrophages; therefore, it may be used to prevent the development of oral cancer.