Frequency and distribution of herpesvirus-like DNA sequences (KSHV) in different stages of classic Kaposi's sarcoma and in normal tissues from an Italian population

The frequency and distribution of herpesvirus-like DNA sequences (KSHV) were investigated by PCR in the pathologic skin lesions of a series of 22 HIV-negative elderly patients with classic Kaposi's sarcoma (KS) from Italy, one of the few regions of the world where classic KS is prevalent. Viral sequences were clearly identifiable in 15 cases, in particular in 2 of 5 patch, in 3 of 6 plaque and in 10 of 11 nodular lesions. Our findings confirm the association of these herpesvirus-like DNA sequences with KS in unrelated populations, providing evidence of the putative KS-associated agent in all different histologic lesions of the disease, mainly in the nodular stage. The search for other herpesviruses by PCR showed that Epstein-Barr virus (EBV) sequences were present in 7 of 22 pathologic skin lesions. In 4 cases, both EBV and KSHV were present. On the contrary, all 22 classic KS specimens were negative for human herpesvirus-6 sequences. Two of 3 patch and the 1 nodular lesions from AIDS-related KS patients examined were positive for KSHV but negative for both EBV and HHV-6 sequences. Furthermore, we evaluated the prevalence of KSHV sequences in the normal population of the same geographical area. Thirteen peripheral blood mononuclear cell samples, 9 salivary gland tissues and 6 saliva samples from healthy subjects were invariably found negative for KSHV, using the same PCR technique. Of interest, 2 of 11 hyperplastic tonsils harboured these herpesvirus-like sequences, suggesting that, like other herpesviruses, the KS-associated agent may be harboured in a proportion of normal individuals and tonsils may represent at least one of the possible reservoirs of this putative lymphotropic γ-herpesvirus in vivo. © 1996 Wiley-Liss, Inc.     

p53 expression and proliferative activity predict survival in non-invasive thymomas

We performed p53 immunohistochemistry, DNA flow cytometry and analysis of the argyrophilic nucleolar organizer regions (AgNORs) in formalin-fixed, paraffin-embedded sections from 46 non-invasive thymomas and correlated the results with the traditional clinicopathologic features of the tumor. p53 immunopositivity was detected in 21 of 46 cases; it was not associated with any clinicopathologic features nor DNA content but significantly correlated with AgNOR counts. On univariate analysis, 10-year survival rates were 100% for p53-negative cases but only 71% for p53-positive cases and 93% for patients with low AgNOR counts but only 77% for patients with high AgNOR counts. Age, sex, histologic type, myasthenia gravis and DNA content did not correlate with survival. Our results indicate that p53 staining and evaluation of proliferative activity allow assessment of prognosis in non-invasive thymomas, when all of the other parameters are insufficient. Furthermore, the high rate of p53 expression in non-invasive thymomas suggests that abnormal p53 immunoreactivity may occur early in the neoplastic process. © 1996 Wiley-Liss, Inc.     

Physical performance in patients with thalassemia before and after transfusion

Patients with thalassemia who are on chronic transfusion programs have chronic ventilatory and cardiocirculatory abnormalities. We studied flow-volume curves, blood gas exchange, and cardiorespiratory responses to exercise in 12 patients with thalassemia major (TM) before and 24 hours after transfusions. Cardiorespiratory fitness was assessed with an exercise tolerance test on a cycle-ergometer. Ten healthy controls underwent the same protocol twice, first at baseline and then 24 hours later, without having had transfusions. We identified two subgroups of patients with a questionnaire: 1) those with no history of airway disease; and 2) those with a history of airway obstruction. Patients with no history of airway disease had normal baseline expiratory flows and no posttransfusion changes; those with a history of airway obstruction had lower pretransfusion expiratory flows rates and significantly decreased posttransfusion forced expiratory volume in 1 second (FEV₁) and forced expiratory flow at 25–75% of forced vital capacity (FEV_25–75%). As a group, TM patients had significantly lower pretransfusion cardiorespiratory function than controls; TM patients' maximum workload was 33% lower, maximum ventilation was 38% lower, maximum oxygen uptake was 25.7% lower, oxygen pulse was 28.6% lower, dyspnea index was 10.6% lower, and ventilatory equivalent for oxygen was 27.1% lower than in control subjects. Although cardiorespiratory responses to exercise improved in both subgroups after transfusion, patients with a history of airways obstruction had a significant posttransfusion increase in their dyspnea index (P = 0.05) and further increased their already abnormally high values of P_ETCO₂ (43 mmHg). These results suggest that the transfusion worsened relative hypoventilation at the maximum workload only in the subgroup with a history of airway obstruction. Pediatr Pulmonol. 1996; 21:367–372. © 1996 Wiley-Liss, Inc.     

Renal vascular damage in systemic sclerosis patients without clinical evidence of nephropathy

Objective. To evaluate the use of color-flow Doppler ultrasonography, a direct, noninvasive technique, for measurement of kidney blood flow in patients with systemic sclerosis (SSc). Methods. Twenty-five normal volunteers and 25 SSc patients (median disease duration 8 years, range 2–21 years) were studied. All were free of clinical symptoms of renal damage. The resistance index (RI) was determined on main, interlobar, and cortical vessels. Results. In SSc patients, the RI was significantly increased at every sampling site examined (P < 0.001). RI values were strongly correlated with disease duration (main artery r = 0.56, P < 0.04; interlobar artery r = 0.63, P < 0.02; cortical artery r = 0.75, P < 0.002). Regression analysis showed no relationship between RI and creatinine clearance values. Conclusion. Color-flow Doppler ultrasonography is a sensitive and noninvasive technique for evaluating vascular damage of the kidney in patients with SSc.     

Surface NMR using quantum sensors in diamond

NMR is a noninvasive, molecular-level spectroscopic technique widely used for chemical characterization. However, it lacks the sensitivity to probe the small number of spins at surfaces and interfaces. Here, we use nitrogen vacancy (NV) centers in diamond as quantum sensors to optically detect NMR signals from chemically modified thin films. To demonstrate the method’s capabilities, aluminum oxide layers, common supports in catalysis and materials science, are prepared by atomic layer deposition and are subsequently functionalized by phosphonate chemistry to form self-assembled monolayers. The surface NV-NMR technique detects spatially resolved NMR signals from the monolayer, indicates chemical binding, and quantifies molecular coverage. In addition, it can monitor in real time the formation kinetics at the solid–liquid interface. With our approach, we show that NV quantum sensors are a surface-sensitive NMR tool with femtomole sensitivity for in situ analysis in catalysis, materials, and biological research.

The characterization of surface processes at the molecular level is essential for understanding fundamental processes in industrial catalysis, energy conversion, electronic circuits, targeted drug delivery, and biosensing (1). However, many analytical techniques used in surface science are inaccessible under ambient or chemically relevant conditions. Therefore, it remains challenging to perform chemical analysis under the conditions in which these processes occur (2, 3). Commonly used surface sensitive methods, such as X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy, and secondary ion mass spectroscopy can perform chemical analysis but require ultra-high vacuum and expensive equipment (4). Great efforts have been devoted to extending XPS analysis to near ambient conditions (2). Indeed, both near-ambient pressure XPS and extended X-ray absorption fine structure have significantly expanded the applicability of these X-ray–based techniques for understanding reaction mechanisms at chemically active interfaces (2, 5). However, both methods require intense synchrotron radiation to achieve high sensitivity and resolution, which limits their practical accessibility and increases their cost. State-of-the-art surface-sensitive spectroscopy techniques, such as sum frequency generation and second harmonic generation, can perform analysis under ambient conditions but require technically complex equipment such as femtosecond lasers (6). Even with all these techniques available, molecular dynamics or chemical reaction kinetics at surfaces are still challenging to probe experimentally (7) (SI Appendix, Supplementary Note 1).NMR spectroscopy is one of the major tools for chemical and structural analysis in chemistry, biology, and materials science. Solid-state NMR in particular (8) has advanced understanding of a range of systems, including metal organic frameworks (9), batteries (10), and catalysts (11). However, sensitivity remains a challenge for traditional NMR spectroscopy, making studies at surfaces difficult because of the limited numbers of nuclear spins. Recently, surface-enhanced NMR spectroscopy (DNP-SENS) relying on hyperpolarization such as dynamic nuclear polarization (12, 13) or xenon-based techniques (14) gained research momentum and enabled probing spins located at surfaces. However, even in highly porous materials with greater than 1,000 m²/g surface area, the concentration of NMR-active nuclei of interest often remains low (e.g., 1 mmol of surface atoms/g), which requires long averaging times to obtain solid-state NMR spectra with reasonable signal-to-noise ratios (SNR) (12) (SI Appendix, Supplementary Note 2).Here, we demonstrate the use of quantum sensors in diamond as a surface-sensitive spectroscopy technique that works at ambient conditions and can probe planar interfaces on the microscopic length scale with far greater sensitivity (femtomoles, see Materials and Methods) than conventional NMR. The spectroscopic technique relies on the nitrogen vacancy (NV) point defect, consisting of a nitrogen impurity (N) and an adjacent vacancy (V) in the carbon lattice of diamond. These spin-1 defects allow for optical detection of magnetic resonance and have been established as highly sensitive nanoscale magnetic field sensors (15, 16). Near-surface NV centers are sensitive to magnetic fields from the Larmor precession of nuclei from samples positioned outside of the diamond. This enables nanoscale NMR detection—even down to a single molecule (17) or spin (18, 19). The measurement volume of such NV sensors (20, 21) corresponds to a hemisphere whose radius is roughly their depth below the surface in the diamond lattice (e.g., 5 to 10 nm). At this small length scale, the thermal polarization of the nuclear spins can be neglected since spin noise dominates for a small number of spins (22, 23). For that reason, the NMR signal strength is independent of the applied magnetic field B₀, reducing experimental complexity and costs, which makes the technique accessible to a broader community. Previously published nanoscale NV-NMR experiments detected NMR signals from either bulk samples [such as viscous oils (21, 22, 24)] or samples tethered to (17) or placed directly on the diamond surface (25). In this work, we propose the use of NV centers in diamond combined with state-of-the-art thin film deposition techniques as a general platform to detect NMR signals with high sensitivity and spatial resolution even from nondiamond surfaces. This approach is general and allows for the probing of a variety of surfaces and interfaces with NMR, thereby enabling their chemistry to be explored. Here, we use atomic layer deposition (ALD), a technology that can be applied to synthesize films of a wide variety of materials with high thickness precision to coat the diamond with amorphous aluminum oxide (Al₂O₃). Al₂O₃ provides an exemplary surface of high technical relevance in optoelectronic applications and acts as structural support in various catalytic processes (26). In a proof-of-concept study for this surface-sensitive spectroscopic technique, we probe the chemical modification of the Al₂O₃ surface with phosphonate anchoring during the formation of a self-assembling monolayer (SAM) (27).