Immune checkpoint expression on peripheral cytotoxic lymphocytes in cervical cancer patients: moving beyond the PD-1/PD-L1 axis.

Immune checkpoint therapy to reverse natural killer (NK) and T cell exhaustion has emerged as a promising treatment in various cancers. While anti-programmed cell death 1 (PD-1) pembrolizumab has recently gained Food and Drug Administration (FDA) approval for use in recurrent or metastatic cervical cancer, other checkpoint molecules, such as T cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibition motif (ITIM) domains (TIGIT) and T cell immunoglobulin and mucin-domain containing-3 (Tim-3), have yet to be fully explored in this disease. We report expression of TIGIT, Tim-3 and PD-1 on subsets of peripheral blood NK (CD56dim/neg CD16bright/dim/neg and CD56bright CD16dim/neg ) and T cells. The percentages of these cells were increased in women with cervical cancer and pre-malignant lesions. PD-1+ NK and T cells were likely to co-express TIGIT and/or Tim-3. These cells, with an apparently 'exhausted' phenotype, were augmented in patients. A subset of cells were also natural killer group 2 member D (NKG2D)- and DNAX accessory molecule 1 (DNAM-1)-positive. PD-1int and PD-1high T cells were notably increased in cervical cancer. Soluble programmed cell death ligand 1 (PD-L1) was higher in cancer patient blood versus healthy donors and we observed a positive correlation between sPD-L1 and PD-1+ T cells in women with low-grade lesions. Within the cancer group, there were no significant correlations between sPD-L1 levels and cervical cancer stage. However, when comparing cancer versus healthy donors, we observed an inverse association between sPD-L1 and total T cells and a correlation between sPD-L1 and CD56dim NK cells. Our results may show an overview of the immune response towards pre-cancerous lesions and cervical cancer, perhaps giving an early clue as to whom to administer blocking therapies. The increase of multiple checkpoint markers may aid in identifying patients uniquely responsive to combined antibody therapies.

[18F]-Sodium fluoride uptake in Takayasu arteritis.

BACKGROUND: While 18F-fluorodeoxyglucose and 18F-sodium fluoride with positron emission tomography relate with inflammation and calcification, their role in the assessment of patients with Takayasu arteritis has not yet been studied. METHODS: We present 5 patients with suspected active metabolic disease who underwent PET with 18F-fluorodeoxyglucose and 18F-sodium fluoride in order to explore the locations and correlations of 18F-fluorodeoxyglucose and 18F-sodium fluoride uptakes. Diagnosis of metabolic active disease was based on 18F-fluorodeoxyglucose uptake. RESULTS: We studied 3 female patients and 2 male patients. Median age was 29 years (min: 19 max: 63). In areas with atherosclerotic plaques, we found a negative correlation between 18F-sodium fluoride and 18F-fluorodeoxyglucose uptakes (r = -0.78) (P = .001). Meanwhile, in areas with only metabolic active disease, we found a positive correlation between 18F-sodium fluoride and 18F-fluorodeoxyglucose uptakes (r = 0.94) (P = .019). CONCLUSIONS: In Takayasu arteritis, 18F-sodium fluoride uptake can document different stages of metabolic disease, even in the absence of active metabolic disease or symptoms.

Serotypes, virulence markers and cell invasion ability of Shiga toxin-producing Escherichia coli strains isolated from healthy dairy cattle.

AIM: The occurrence of virulence markers, serotypes and invasive ability were investigated in Shiga toxin-producing Escherichia coli (STEC) isolated from faecal samples of healthy dairy cattle at Rio de Janeiro State, Brazil. METHODS AND RESULTS: From 1562 stx-positive faecal samples, 105 STEC strains were isolated by immuno-magnetic separation (IMS) or plating onto MacConkey agar (MC) followed by colony hybridisation. Fifty (47·6%) strains belonged to nine serotypes (O8:H19, O22:H8, O22:H16, O74:H42, O113:H21, O141:H21, O157:H7, O171:H2 and ONT:H21). The prevalent serotypes were O157:H7 (12·4%), O113:H21 (6·7%) and O8:H19 (5·7%). Virulence genes were identified by polymerase chain reaction (PCR). E-hlyA (77·1%) was the more prevalent virulence marker, followed by espP (64·8%), saa (39%), eae (24·8%) and astA (21·9%). All O157:H7 strains carried the γ (gamma) variant of the locus of enterocyte effacement (LEE) genes and the stx2c gene, while the stx1/stx2 genotype prevailed among the eae-negative strains. None of the eae-positive STEC produced the localized adherence (LA) phenotype in HEp-2 or Caco-2 cells. However, intimate attachment (judged by the fluorescent actin staining test) was detected in some eae-positive strains, both in HEp-2 (23·1%) and in Caco-2 cells (11·5%). Most strains (87·5%) showed 'peripheral association' (PA) adherence phenotype to undifferentiated Caco-2 cells. Twenty-five (92·6%) of 27 strains invaded Caco-2 cells. The highest average value of invasion (9·6%) was observed among the eae-negative bovine strains from serotypes described in human disease. CONCLUSION: Healthy dairy cattle is a reservoir of STEC carrying virulence genes and properties associated with human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: Although reports of human disease associated with STEC are scarce in Brazil, the colonization of the animal reservoir by potentially pathogenic strains offers a significant risk to our population.

Retina layer segmentation using kernel graph cuts and continuous max-flow.

Circular scan Spectral-Domain Optic Coherence Tomography imaging (SD-OCT) is one of the best tools for diagnosis of retinal diseases. This technique provides more comprehensive detail of the retinal morphology and layers around the optic disc nerve head (ONH). Since manual labelling of the retinal layers can be tedious and time consuming, accurate and robust automated segmentation methods are needed to provide the thickness evaluation of these layers in retinal disorder assessments such as glaucoma. The proposed method serves this purpose by performing the segmentation of retinal layers boundaries in circular SD-OCT scans acquired around the ONH. The layers are detected by adapting a graph cut segmentation technique that includes a kernel-induced space and a continuous multiplier based max-flow algorithm. Results from scan images acquired with Spectralis (Heidelberg Engineering, Germany) prove that the proposed method is robust and efficient in detecting the retinal layers boundaries in images. With a mean root-mean-square error (RMSE) of 0.0835 ± 0.0495 and an average Dice coefficient of 0.9468 ± 0.0705 pixels for the retinal nerve fibre layer thickness, the proposed method demonstrated effective agreement with manual annotations.

Hierarchical nanoparticle ensembles synthesized by liquid phase directed self-assembly.

A liquid metal filament supported on a dielectric substrate was directed to fragment into an ordered, mesoscale particle ensemble. Imposing an undulated surface perturbation on the filament forced the development of a single unstable mode from the otherwise disperse, multimodal Rayleigh-Plateau instability. The imposed mode paved the way for a hierarchical spatial fragmentation of the filament into particles, previously seen only at much larger scales. Ultimately, nanoparticle radius control is demonstrated using a micrometer scale switch.

Assessing the influence of topography and canopy structure on Douglas fir throughfall with LiDAR and empirical data in the Santa Cruz mountains, USA.

Atmospheric inputs to forest ecosystems vary considerably over small spatial scales due to subtle changes in relief and vegetation structure. Relationships between throughfall fluxes (ions that pass through the canopy in water), topographic and canopy characteristics derived from sub-meter resolution light detection and ranging (LiDAR), and field measurements were compared to test the potential utility of LiDAR in empirical models of atmospheric deposition. From October 2012 to May 2013, we measured bulk (primarily wet) deposition and sulfate-S, chloride (Cl(-)), and nitrate-N fluxes beneath eight clusters of Douglas fir trees differing in size and canopy exposure in the Santa Cruz Mountains, California. For all trees sampled, LiDAR data were used to derive canopy surface height, tree height, slope, and canopy curvature, while tree height, diameter (DBH), and leaf area index were measured in the field. Wet season throughfall fluxes to Douglas fir clusters ranged from 1.4 to 3.8 kg S ha(-1), 17-54 kg Cl(-) ha(-1), and 0.2-4 kg N ha(-1). Throughfall S and Cl(-) fluxes were highest under clusters with large trees at topographically exposed sites; net fluxes were 2-18-fold greater underneath exposed/large clusters than all other clusters. LiDAR indices of canopy curvature and height were positively correlated with net sulfate-S fluxes, indicating that small-scale canopy surface features captured by LiDAR influence fog and dry deposition. Although tree diameter was more strongly correlated with net sulfate-S throughfall flux, our data suggest that LiDAR data can be related to empirical measurements of throughfall fluxes to generate robust high-resolution models of atmospheric deposition.

Effect of Dunaliella tertiolecta organic exudates on the Fe(II) oxidation kinetics in seawater.

The role played by the natural organic ligands excreted by the green algae Dunaliella tertiolecta on the Fe(II) oxidation rate constants was studied at different stages of growth. The concentration of dissolved organic carbon increased from 2.1 to 7.1 mg L(-1) over time of culture. The oxidation kinetics of Fe(II) was studied at nanomolar levels and under different physicochemical conditions of pH (7.2-8.2), temperature (5-35 °C), salinity (10-37), and dissolved organic carbon produced by cells (2.1-7.1 mg L(-1)). The experimental rate always decreased in the presence of organic exudates with respect to that in the control seawater. The Fe(II) oxidation rate constant was also studied in the context of Marcus theory, where ΔG° was 39.31-51.48 kJ mol(-1). A kinetic modeling approach was applied for computing the equilibrium and rate constants for Fe(II) and exudates present in solution, the Fe(II) speciation, and the contribution of each Fe(II) species to the overall oxidation rate constant. The best fit model took into account two acidity equilibrium constants for the Fe(II) complexing ligands with pKa,1=9.45 and pKa,2=4.9. The Fe(II) complexing constants were KFe(II)-LH=3×10(10) and KFe(II)-L=10(7), and the corresponding computed oxidation rates were 68±2 and 36±8 M(-1) min(-1), respectively.

A multiple hollow fibre liquid-phase microextraction method for the determination of halogenated solvent residues in olive oil.

The present paper describes a method based on the extraction of analytes by multiple hollow fibre liquid-phase microextraction and detection by ion-trap mass spectrometry and electron capture detectors after gas chromatographic separation. The limits of detection are in the range of 0.13-0.67 µg kg(-1), five orders of magnitude lower than those reached with the European Commission Official method of analysis, with three orders of magnitude of linear range (from the quantification limits to 400 µg kg(-1) for all the analytes) and recoveries in fortified olive oils in the range of 78-104 %. The main advantages of the analytical method are the absence of sample carryover (due to the disposable nature of the membranes), high enrichment factors in the range of 79-488, high throughput and low cost. The repeatability of the analytical method ranged from 8 to 15 % for all the analytes, showing a good performance.

Global decrease in heavy metal concentrations in brown algae in the last 90 years.

In the current scenario of global change, heavy metal pollution is of major concern because of its associated toxic effects and the persistence of these pollutants in the environment. This study is the first to evaluate the changes in heavy metal concentrations worldwide in brown algae over the last 90 years (>15,700 data across the globe reported from 1933 to 2020). The study findings revealed significant decreases in the concentrations of Cd, Co, Cr, Cu, Fe, Hg, Mn, Pb and Zn of around 60-84% (ca. 2% annual) in brown algae tissues. The decreases were consistent across the different families considered (Dictyotaceae, Fucaceae, Laminariaceae, Sargassaceae and Others), and began between 1970 and 1990. In addition, strong relationships between these trends and pH, SST and heat content were detected. Although the observed metal declines could be partially explained by these strong correlations, or by adaptions in the algae, other evidences suggest an actual reduction in metal concentrations in oceans because of the implementation of environmental policies. In any case, this study shows a reduction in metal concentrations in brown algae over the last 50 years, which is important in itself, as brown algae form the basis of many marine food webs and are therefore potential distributors of pollutants.

Imaging the first-order magnetic transition in La0.35Pr0.275Ca0.375MnO3.

The nature of the ferromagnetic, charge, orbital, and antiferromagnetic order in La0.35Pr0.275Ca0.375MnO3 on the nano- and microscale was investigated by photoemission electron microscopy (PEEM) and resonant elastic soft x-ray scattering (RSXS). The structure of the ferromagnetic domains around the Curie temperature T(C) indicates that they nucleate under a high degree of lattice strain, which is brought about by the charge, orbital, and antiferromagnetic order. The combined temperature-dependent PEEM and RSXS measurements suggest that the lattice distortions associated with charge and orbital order are glassy in nature and that phase separation is driven by the interplay between it and the more itinerant charge carriers associated with ferromagnetic metallic order, even well below T(C).

Ferromagnetic enhancement of CE-type spin ordering in (Pr,Ca)MnO3.

We present resonant soft x-ray scattering results from small bandwidth manganites (Pr,Ca)MnO(3), which show that the CE-type spin ordering (SO) at the phase boundary is stabilized only below the canted antiferromagnetic transition temperature and enhanced by ferromagnetism in the macroscopically insulating state (FM-I). Our results reveal the fragility of the CE-type ordering that underpins the colossal magnetoresistance effect in this system, as well as an unexpected cooperative interplay between FM-I and CE-type SO which is in contrast to the competitive interplay between the ferromagnetic metallic state and CE-type ordering.

Competing liquid phase instabilities during pulsed laser induced self-assembly of copper rings into ordered nanoparticle arrays on SiO2.

Nanoscale copper rings of different radii, thicknesses, and widths were synthesized on silicon dioxide thin films and were subsequently liquefied via a nanosecond pulse laser treatment. During the nanoscale liquid lifetimes, the rings experience competing retraction dynamics and thin film and/or Rayleigh-Plateau types of instabilities, which lead to arrays of ordered nanodroplets. Surprisingly, the results are significantly different from those of similar experiments carried out on a Si surface. We use hydrodynamic simulations to elucidate how the different liquid/solid interactions control the different instability mechanisms in the present problem.

Storage and recycling of major and trace element in mangroves.

The role of mangroves in sequestering metal and nutrients in sediment has been described in the past, but knowledge gaps still exist on storage capacity and recycling fluxes of elements in plant biomass, notably concerning their magnitude in root uptake and loss by litterfall. This study addresses the storage and transport pathways of 16 elements, classified as macro-nutrients (Ca, Mg, Na, K), micro-nutrients (Fe, Mn, Ni, Co, Cu, Cr, Zn, Mo), and potential toxicants (Al, Cd, Sn, Pb) in the world's largest mangroves, the Sundarbans. Elemental concentrations in plant organs were generally lower than in the sediment. The stock of macro and micro-nutrients in plant biomass varied from 60 to 2717 and 0.003 to 37.7 Mg ha-1 respectively, with highest values observed for Na and lowest for Cd. The Avicennia species exhibited the maximal accumulation of all elements. Translocation of major elements to different plant organs increased with increasing their concentrations in the sediment. Elemental loss via litterfall indicated that Sundarbans mangrove could act as a source, particularly of Mn, to the Bay of Bengal. Moreover, belowground uptake of the 16 elements showed 2-3 fold higher fluxes than their loss via litterfall. There was a significant retention of some trace elements (notably Mo, Cd, and Sn) in plant biomass, which might allow one to use these mangroves for phytoremediation and restoration purposes. We conclude that mangroves efficiently store and remobilize major and trace elements from the sediments by root uptake and recycle back to sediment surface via litterfall.

CO2 fluxes in the Northeast Atlantic Ocean based on measurements from a surface ocean observation platform.

The seasonal and spatial variability of the CO2 system parameters and CO2 air-sea exchange were studied in the Northeast Atlantic Ocean between the northwest African coastal upwelling and the oligotrophic open-ocean waters of the North Atlantic subtropical gyre. Data was collected aboard a volunteer observing ship from February 2019 to February 2020. The seasonal and spatial variability of CO2 fugacity in seawater (fCO2,sw) was strongly driven by the seasonal temperature variation, which increased with latitude and was lower throughout the year in coastal regions where the upwelling and offshore transport was more intense. The thermal to biological effect ratio (T/B) was approximately 2, with minimum values along the African coastline related to higher biological activity in the upwelled waters. The fCO2,sw increased from winter to summer by 11.84 ± 0.28 µatm°C-1 on the inter-island routes and by 11.71 ± 0.25 µatm°C-1 along the northwest African continental shelf. The seasonality of total inorganic carbon normalized to constant salinity of 36.7 (NCT) was studied throughout the region. The effect of biological processes and calcification/dissolution on NCT between February and October represented >90% of the reduction of inorganic carbon while air-sea exchange described <6%. The seasonality of air-sea CO2 exchange was controlled by temperature. The surface waters of the entire region acted as a CO2 sink during the cold months and as a CO2 source during the warm months. The Canary basin acted as a net sink of -0.26 ± 0.04 molC m-2 yr-1. The northwest African continental shelf behaved as a stronger sink at -0.48 ± 0.09 molC m-2 yr-1. The calculated average CO2 flux for the entire area was -2.65 ± 0.44 TgCO2 yr-1 (-0.72 ± 0.12 TgC yr-1).

Tropical land-cover change alters biogeochemical inputs to ecosystems in a Mexican montane landscape.

In tropical regions, the effects of land-cover change on nutrient and pollutant inputs to ecosystems remain poorly documented and may be pronounced, especially in montane areas exposed to elevated atmospheric deposition. We examined atmospheric deposition and canopy interactions of sulfate-sulfur (SO4(2-)-S), chloride (Cl-), and nitrate-nitrogen (NO(3-)-N) in three extensive tropical montane land-cover types: clearings, forest, and coffee agroforest. Bulk and fog deposition to clearings was measured as well as throughfall (water that falls through plant canopies) ion fluxes in seven forest and five coffee sites. Sampling was conducted from 2005 to 2008 across two regions in the Sierra Madre Oriental, Veracruz, Mexico. Annual throughfall fluxes to forest and coffee sites ranged over 6-27 kg SO4(2-)-S/ha, 12-69 kg Cl-/ha, and 2-6 kg NO(3-)-N/ha. Sulfate-S in forest and coffee throughfall was higher or similar to bulk S deposition measured in clearings. Throughfall Cl- inputs, however, were consistently higher than Cl- amounts deposited to cleared areas, with net Cl- fluxes enhanced in evergreen coffee relative to semi-deciduous forest plots. Compared to bulk nitrate-N deposition, forest and coffee canopies retained 1-4 kg NO(3-)-N/ha annually, reducing NO(3-)-N inputs to soils. Overall, throughfall fluxes were similar to values reported for Neotropical sites influenced by anthropogenic emissions, while bulk S and N deposition were nine- and eightfold greater, respectively, than background wet deposition rates for remote tropical areas. Our results demonstrate that land-cover type significantly alters the magnitude and spatial distribution of atmospheric inputs to tropical ecosystems, primarily through canopy-induced changes in fog and dry deposition. However, we found that land cover interacts with topography and climate in significant ways to produce spatially heterogeneous patterns of anion fluxes, and that these factors can converge to create deposition hotspots. For land managers, this finding suggests that there is potential to identify species and ecosystems at risk of excess and increasing deposition in montane watersheds undergoing rapid transformation. Our data further indicate that montane ecosystems are vulnerable to air pollution impacts in this and similar tropical regions downwind of urban, industrial, and agricultural emission sources.

Ergonomic assessment of a new hand tool design for laparoscopic surgery based on surgeons' muscular activity.

Laparoscopic surgery techniques are customarily used in non-invasive procedures. That said traditional surgical instruments and devices used by surgeons suffer from certain ergonomic deficiencies that may lead to physical complaints in upper limbs and back and general discomfort that may, in turn, affect the surgeon's skills during surgery. A novel design of the laparoscopic gripper handle is presented and compared with one of the most used instruments in this field in an attempt to overcome this problem. The assessment of the ergonomic feature of the novel design was performed by using time-frequency analysis of the surface electromyography (sEMG) signal during dynamic activities. Singular Spectrum Analysis (SSA) was used to decompose the sEMG signal and extract the median frequency of each muscle to assess muscle fatigue. The results reveal that using the proposed ergonomic grip reduces the mean values of the muscle activity during each of the proposed tasks. The novel design also improves the ease of use in laparoscopic surgery as it minimises high-pressure contact areas, reduces large amplitude movements and promotes a neutral position of the hand, wrist and forearm. Furthermore, the SSA method for time-frequency analysis provides a powerful tool to analyse a prescribed activity in ergonomic terms. The proposed methodology to assess muscle activity during surgery activities may be useful in the selection of surgical instruments when programming extended procedures, as it provides an additional selection criterion based on the surgeon's biomechanics and the proposed activity.

Optimization and validation of headspace sorptive extraction for the analysis of volatile compounds in wine vinegars.

Quantification of aroma compounds in wine vinegars is challenging due to the complexity of the matrix and the low concentrations expected. A method for the determination of volatile compounds in wine vinegars employing headspace sorptive extraction-thermal desorption-gas chromatography-mass spectrometry (HSSE-TD-GC-MS) was developed. A central composite design was used to optimize the sampling condition. The proposed method was successfully validated and low detection and quantification limits was obtained. The application of the proposed methodology allows the determination of 53 compounds in different wine vinegars (red, Sherry). Five of them have been detected in wine vinegars for the first time.

Genetic relatedness of a non-motile variant O157 enteropathogenic Escherichia coli (EPEC) strain and E. coli strains belonging to pathogenic related groups.

The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.

Repeated red wine consumption and changes on plasma antioxidant capacity and endogenous antioxidants (uric acid and protein thiol groups).

This study aims to ascertain the in vivo antioxidant properties of red wine by determining how it affects antioxidant biomarkers (plasma antioxidant capacity (PAC) and endogenous antioxidants such as uric acid or protein thiol groups). Antioxidant biomarkers have been assessed in eight healthy human volunteers after repeated intakes of 300 mL of red wine every day for a week. During this intervention period, volunteers followed a low phenolic diet designed to prevent the phenolic compounds in wine from interfering with the phenolics from other foods or beverages. This diet was followed throughout the week that the volunteers drank wine and for another control week when they drank water. Biomarkers were determined before the subjects taking part in the study started the intervention period with red wine (Monastrell variety) and 1, 3, and 7 days after. PAC was evaluated by the Ferric Reducing Ability of Plasma assay (FRAP), and the Oxygen Radical Absorbance Capacity assay using fluorescein (ORAC-FL). In addition, the concentrations of endogenous antioxidants such as uric acid, albumin, bilirubin, and protein thiol groups were analyzed. The FRAP method shows that PAC increased after the week of wine consumption but decreased after the week without wine consumption. The uric acid concentration did not show any changes that were significantly different from our results in acute wine intake studies. Protein thiol groups decreased significantly (p < 0.05) with the low phenolic diet, but this decrease was not statistically significant if the diet was taken with red wine (p < 0.05).

Stability study of a constant-volume thin film flow.

We study the stability of a constant volume of fluid spreading down an incline. In contrast to the commonly considered flow characterized by constant fluid flux, in the present problem the base flow is time dependent. We present a method to carry out consistently linear stability analysis, based on simultaneously solving the time evolution of the base flow and of the perturbations. The analysis is performed numerically by using a finite-difference method supplemented with an integral method developed here. The computations show that, after a short transient stage, imposed perturbations travel with the same velocity as the leading contact line. The spectral analysis of the modes evolution shows that their growth rates are, in general, time dependent. The wavelength of maximum amplitude, lambda_{max} , decreases with time until it reaches an asymptotic value which is in good agreement with experimental results. We also explore the dependence of lambda_{max} on the cross sectional fluid area A , and on the inclination angle alpha of the substrate. For considered small A 's, corresponding to small Bond numbers, we find that the dependence of lambda_{max} on A is in good agreement with experimental data. This dependence differs significantly from the one observed for the films characterized by much larger A 's and Bond numbers. We also predict the dependence of lambda_{max} on the inclination angle alpha .