Penicillium polonicum-mediated green synthesis of silver nanoparticles: Unveiling antimicrobial and seed germination advancements.

Silver nanoparticles (AgNPs), widely recognized for their nanoscale geometric size and unique properties, such as large specific surface area, high permeability, and high safety, were synthesized using the endophytic fungus Penicillium polonicum PG21 through a green approach. Four key synthesis factors-48 h, 45 °C, pH 9.0, and 80 mM AgNPs concentration-were optimized. Characterization via ultraviolet-visible spectroscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, and X-ray diffraction revealed the AgNPs as approximately 3-25 nm spherical particles with numerous functional groups ensuring stability. AgNPs were tested against various fungal and bacterial plant pathogens, including Botrytis cinerea (EB-1), Alternaria alternata (EB-2, EB-3), Fusarium solani (RG-1), Williamsia serinedens (SL-1), Sphingopyxis macrogoltabida (SL-2), Bacillus velezensis (SL-3), and Pseudomonas mediterranea (SL-4), causing agricultural challenges. PG21-synthesized AgNPs exhibited inhibition rates against all tested fungi, with 60 µg/mL AgNPs demonstrating optimal inhibition rates. Notably, EB-1 experienced a significant growth inhibition, reaching an inhibition rate reached of 74.22 ± 1.54%. Conversely, RG-1 exhibited the smallest inhibitory effect at 48.13 ± 0.92%. The effect of AgNPs on safflower seed germination and growth revealed notable increases in shoot length, fresh weight, stem length, and number of lateral roots-1.4, 1.4, 1.33, and 10.67 times higher than the control, respectively, at an AgNPs concentration of 80 µg/mL. In conclusion, green-synthesized AgNPs demonstrate pathogen toxicity, showcasing potential applications in disease management for industrial crops and promoting plant growth.

On the imaging depth limit of photoacoustic tomography in the visible and first near-infrared windows.

It is well known that photoacoustic tomography (PAT) can circumvent the photon scattering problem in optical imaging and achieve high-contrast and high-resolution imaging at centimeter depths. However, after two decades of development, the long-standing question of the imaging depth limit of PAT in biological tissues remains unclear. Here we propose a numerical framework for evaluating the imaging depth limit of PAT in the visible and the first near-infrared windows. The established framework simulates the physical process of PAT and consists of seven modules, including tissue modelling, photon transportation, photon to ultrasound conversion, sound field propagation, signal reception, image reconstruction, and imaging depth evaluation. The framework can simulate the imaging depth limits in general tissues, such as the human breast, the human abdomen-liver tissues, and the rodent whole body and provide accurate evaluation results. The study elucidates the fundamental imaging depth limit of PAT in biological tissues and can provide useful guidance for practical experiments.

Photoacoustic imaging of squirrel monkey cortical responses induced by peripheral mechanical stimulation.

Non-human primates (NHPs) are crucial models for studies of neuronal activity. Emerging photoacoustic imaging modalities offer excellent tools for studying NHP brains with high sensitivity and high spatial resolution. In this research, a photoacoustic microscopy (PAM) device was used to provide a label-free quantitative characterization of cerebral hemodynamic changes due to peripheral mechanical stimulation. A 5 × 5 mm area within the somatosensory cortex region of an adult squirrel monkey was imaged. A deep, fully connected neural network was characterized and applied to the PAM images of the cortex to enhance the vessel structures after mechanical stimulation on the forelimb digits. The quality of the PAM images was improved significantly with a neural network while preserving the hemodynamic responses. The functional responses to the mechanical stimulation were characterized based on the improved PAM images. This study demonstrates capability of PAM combined with machine learning for functional imaging of the NHP brain.

Acceleration of Protein Degradation by 20S Proteasome-Binding Peptides Generated by In Vitro Artificial Evolution.

Although the 20S core particle (CP) of the proteasome is an important component of the 26S holoenzyme, the stand-alone 20S CP acts directly on intrinsically disordered and oxidized/damaged proteins to degrade them in a ubiquitin-independent manner. It has been postulated that some structural features of substrate proteins are recognized by the 20S CP to promote substrate uptake, but the mechanism of substrate recognition has not been fully elucidated. In this study, we screened peptides that bind to the 20S CP from a random eight-residue pool of amino acid sequences using complementary DNA display an in vitro molecular evolution technique. The identified 20S CP-binding amino acid sequence was chemically synthesized and its effects on the 20S CP were investigated. The 20S CP-binding peptide stimulated the proteolytic activity of the inactive form of 20S CP. The peptide bound directly to one of the α-subunits, opening a gate for substrate entry on the α-ring. Furthermore, the attachment of this peptide sequence to α-synuclein enhanced its degradation by the 20S CP in vitro. In addition to these results, docking simulations indicated that this peptide binds to the top surface of the α-ring. These peptides could function as a key to control the opening of the α-ring gate.

In vivo monitoring of hemodynamic changes in ischemic stroke using photoacoustic tomography.

Ischemic stroke occurs when a blood vessel supplying the brain is blocked, leading to decreased blood flow. Early diagnosis and treatment are crucial. However, existing clinical imaging methods have limitations, such as safety issues and low time resolution. To address these challenges, we propose using photoacoustic tomography (PAT) with a contrast agent, known for its high resolution and contrast capabilities. Our study involved imaging brain vasculature in three groups: normal, unilateral common carotid artery ligation (UCAL), and middle cerebral artery occlusion (MCAO). On the ischemic stroke side, we observed reduced blood vessel density and hemodynamic changes were evident after injecting indocyanine green for PAT. The photoacoustic intensity was notably lower in the ligated sides of the UCAL and MCAO groups, with statistically significant differences between the three groups. This work highlights PAT's potential as a powerful tool for early diagnosis and guidance in ischemic stroke cases.

Multibeam minimum variance beamforming for ring array ultrasound imaging.

Objective. Delay-and-sum (DAS) and minimum variance (MV) are two of the most important beamformers researched in ultrasound imaging. Compared with DAS, MV beamformer is different in respect of the aperture weights calculation, and can enhance the image quality by minimizing interference signal power. Various MV beamformers in linear array are studied, but linear array only provides a limited field of view. Ring array can provide better resolution and a full viewing angle; however, few studies have been explored based on ring array transducers.Approach. In this study, we proposed the multibeam MV (MB-MV) beamformer based on the conventional MV to enhance the image quality in ring array ultrasound imaging. To assess the effectiveness of the proposed approach, we conducted simulations, phantom experiments, andin vivohuman experiments to compare MB-MV with DAS and spatial smoothing (SS) MV beamformers.Main results. The results show that the MB-MV method achieves at least 50% enhancement in terms of full width at half maximum compared to the others. Additionally, the MB-MV method improves the contrast ratio by approximate 6 dB and 4 dB compared with DAS and SS MV, respectively.Significance. This work demonstrates the feasibility of MB-MV method for ring array ultrasound imaging, and proves that MB-MV can improve the imaging quality in medical ultrasound imaging. According to our results, MB-MV method provides great potential in distinguishing between lesion and non-lesion areas in clinics, and further promotes the practical application of ring arrays in ultrasound imaging.

Enolase, a cadmium resistance related protein from hyperaccumulator plant Phytolacca americana, increase the tolerance of Escherichia coli to cadmium stress.

Phytolacca americana is a Cd hyperaccumulator plant that accumulates significant amounts of Cd in leaves, making it a valuable phytoremediation plant species. Our previous research found enolase (ENO) may play an important part in P. americana to cope with Cd stress. As a multifunctional enzyme, ENO was involved not only in glycolysis but also in the response of plants to various environmental stresses. However, there are few studies on the function of PaENO (P. americana enolase) in coping with Cd stress. In this study, the PaENO gene was isolated from P. americana, and the expression level of PaENO gene significantly increased after Cd treatment. The enzymatic activity analysis showed PaENO had typical ENO activity, and the 42-position serine was essential to the enzymatic activity of PaENO. The Cd resistance assay indicated the expression of PaENO remarkably enhanced the resistance of E. coli to Cd, which was achieved by reducing the Cd content in E. coli. Moreover, both the expression of inactive PaENO and PaMBP-1 (alternative translation product of PaENO) can improve the tolerance of E. coli to Cd. The results indicated PaENO may be alternatively translated into the transcription factor PaMBP-1 to participate in the response of P. americana to Cd stress.

The expression of the Cd resistance related protein PaENO can significantly increase the tolerance of E. coli to Cd, which was achieved by reducing the content of Cd in E. coli cells, and was independent of the enzymatic activity of PaENO. Moreover, PaENO may be alternatively translated into the transcription factor PaMBP-1 to participate in the response of P. americana to Cd stress.

Small-core hollow-core nested antiresonant nodeless fiber with semi-circular tubes.

Hollow-core nested anti-resonant nodeless fibers (HC-NANFs) exhibit great performance in low loss and large bandwidth. Large core sizes are usually used to reduce confinement losses, but meanwhile, bring side effects such as high bending and coupling losses. This study proposes a small-core HC-NANF with a relatively low confinement loss. Semi-circular tubes (SCTs) are added to constitute the core boundary and reduce the fiber-core radius (R). Double NANFs tubes and single-ring tubes are added inside the SCTs to reduce loss. Simulation results show that the optimized structure with R of 5 µm has confinement loss and total loss of 0.687 dB/km and 4.27 dB/km at 1.55 µm, respectively. The bending loss is less than 10 dB/km at 1.4 ∼ 1.6 µm with a bending radius of 10 mm. The direct coupling loss with standard single mode fiber is greatly reduced to ∼ 0.125 dB compared to other HC-NANFs. The modified structure of HC-NANFs also shows a large bandwidth, effective single-mode operation, potentially high birefringence performance, and remarkable robustness of the optimized structure parameters, making it suitable for short-haul applications in laser-based gas sensing, miniaturized fiber sensing, etc.

From Plant to Yeast-Advances in Biosynthesis of Artemisinin.

Malaria is a life-threatening disease. Artemisinin-based combination therapy (ACT) is the preferred choice for malaria treatment recommended by the World Health Organization. At present, the main source of artemisinin is extracted from Artemisia annua; however, the artemisinin content in A. annua is only 0.1-1%, which cannot meet global demand. Meanwhile, the chemical synthesis of artemisinin has disadvantages such as complicated steps, high cost and low yield. Therefore, the application of the synthetic biology approach to produce artemisinin in vivo has magnificent prospects. In this review, the biosynthesis pathway of artemisinin was summarized. Then we discussed the advances in the heterologous biosynthesis of artemisinin using microorganisms (Escherichia coli and Saccharomyces cerevisiae) as chassis cells. With yeast as the cell factory, the production of artemisinin was transferred from plant to yeast. Through the optimization of the fermentation process, the yield of artemisinic acid reached 25 g/L, thereby producing the semi-synthesis of artemisinin. Moreover, we reviewed the genetic engineering in A. annua to improve the artemisinin content, which included overexpressing artemisinin biosynthesis pathway genes, blocking key genes in competitive pathways, and regulating the expression of transcription factors related to artemisinin biosynthesis. Finally, the research progress of artemisinin production in other plants (Nicotiana, Physcomitrella, etc.) was discussed. The current advances in artemisinin biosynthesis may help lay the foundation for the remarkable up-regulation of artemisinin production in A. annua through gene editing or molecular design breeding in the future.

Prototype endoscopic photoacoustic-ultrasound balloon catheter for characterizing intestinal obstruction.

In our previous studies, we have demonstrated the feasibility of characterizing intestinal inflammation and fibrosis using endoscopic photoacoustic imaging. Purposed at te clinical translation of the imaging technology, we developed a photoacoustic/ultrasound imaging probe by integrating a miniaturized ultrasound array and an angle-tipped optical fiber in a hydrostatic balloon catheter. When collapsed, the catheter probe may potentially be compatible with a clinical ileo-colonoscope. In addition, the flexible surface of the hydrostatic balloon allows for acoustic coupling at the uneven surfaces of the gas-filled intestine. Tissue phantom studies show that the catheter probe possesses an imaging penetration of at least 12 mm. Experiments with a rabbit model in vivo validated the probe in differentiating normal, acute and chronic conditions in intestinal obstruction.

Genome-wide identification and characterization of the HD-Zip gene family and expression analysis in response to stress in Rehmannia glutinosa Libosch.

The HD-Zip family of transcription factors is unique to the plant kingdom, and play roles in modulation of plant growth and response to environmental stresses. R. glutinosa is an important Chinese medicinal material. Its yield and quality are susceptible to various stresses. The HD-Zip transcription factors is unique to the plant, and roles in modulation of plant growth and response to environmental stresses. However, there is no relevant research on the HD-ZIP of R. glutinosa. In this study, 92 HD-Zip transcription factors were identified in R. glutinosa, and denominated as RgHDZ1-RgHDZ92. Members of RgHDZ were classified into four groups (HD-ZipI-IV) based on the phylogenetic relationship of Arabidopsis HD-Zip proteins, and each group contains 38, 18, 17, and 19 members, respectively. Expression analyses of RgHDZ genes based on transcriptome data showed that the expression of these genes could be induced by the endophytic fungus of R. glutinosa. Additionally, we showed that RgHDZ genes were differentially expressed in response to drought, waterlogging, temperature, and salinity treatments. This study provides important information for different expression patterns of stress-responsive HD-Zip and may contribute to the better understanding of the different responses of plants to biotic and abiotic stresses, and provide a molecular basis for the cultivation of resistant varieties of R. glutinosa.

Prognostic factors and related complications/sequalae of squamous cell carcinoma located in the gingivobuccal complex.

BACKGROUND: Gingivobuccal complex (GBC) was a relatively new concept of oral subsite that was comprises of the upper and/or lower gingiva, gingival buccal sulcus, and adjacent buccal mucosa. Squamous cell carcinoma (SCC) of the GBC had a poor prognosis, with few studies analyzing this particular entity. The objective of this study was to analyze the risk factors affecting the prognosis and complications/sequalae of gingivobuccal complex cancer. METHODS: Between December 2014 and August 2019, a total of 122 patients diagnosed with primary gingivobuccal complex cancer in Beijing Stomatological Hospital, Capital Medical University were enrolled in the study. Through outpatient reviewed and telephone followed-up for 2-5 years postoperatively, postoperative relapse and complications/sequalae were assessed. The primary outcome parameter was 2-year disease-free survival. RESULTS: The most common central site of the tumor was the buccal mucosa (45.1%), followed by the lower gingiva (36.9%). The most diseases were pT4a (45.1%) and there was lymph node invasion (pN+) in 41.8% of patients. Moderate differentiated squamous carcinoma (77.9%) accounted for the vast majority of the histopathological differentiation. A total of 62.3% of tumors invaded the bone, while, 5.7% invaded the skin layer. Survival analysis found that 44.3% of patients experienced relapse within two years postoperatively and the mortality rate after relapse was 75.9%. Almost 60.0% of the tumors involving the maxilla and/or mandible developed relapse. Cox proportional hazards model found that pN stage (p= 0.002) and bone invasion (p= 0.007) were significant independent predictors of 2-year disease-free survival. Importantly, 63.1% of patients had postoperative (and postradiotherapy) complications/sequalae. It was noteworthy that 18 of 43 patients (41.9%) who implanted with titanium plates had hardware-related complications/sequalae, and the most of them were titanium plate exposure (61.1%). CONCLUSIONS: Squamous cell carcinoma of the gingivobuccal complex cancer, as a new subsite worthy of attention in oral cancer, has a high complication/sequalae rate, high relapse rate and poor prognosis. TRIAL REGISTRATION: Prospective, Observational, Real-world Oral Malignant Tumors Study ( clinicaltrials.gov identifier: NCT02395367). The approval of the Institutional Review Board of the Beijing Stomatological Hospital of Capital Medical University (Approval number: CMUSH-IRB-KJPJ-2015-08).

[Identification of miR160 family and their target genes in Rehmannia glutinosa in response to endophytic fungus GG22 infection].

This study aims to reveal the possible role of miR160 family in Rehmannia glutinosa in response to the infection of endophytic fungus Fusarium oxysporum GG22. Specifically, miR160 precursors and mature miR160 were retrieved from the small RNA database yielded by high-throughput sequencing. RNAfold was used to analyze the precursor structure, and DNAMAN and MEGA to analyze conservation and evolution of miR160 precursors and mature miR160. The target genes of miR160 were predicted and annotated, and the interaction was analyzed. Based on degradome sequencing, the target genes were further identified. The results showed that miR160 precursors had intact stem-loop structures. The precursor and mature sequences were conserved, particularly the 3 rd-16 th bases of the 5'-terminal. According to the phylogenetic tree, R. glutinosa had close evolutionary relationship with Arabidopsis thaliana, Oryza sativa, Salvia miltiorrhiza, and Sesamum indicum. A total of 22 target genes of miR160 were predicted and most of them were auxin response factor(ARF) genes. The target genes were involved in the Gene Ontology(GO) terms of biological processes, cellular components, and molecular functions. According to the degradome sequencing results, four target genes of miR160 were ARF(ARF18, ARF22) genes. R. glutinosa regulated its growth in response to the infection of endophytic fungus by changing the expression of miR160 and the target genes. qRT-PCR result of the differentially expressed rgl-miR160a and rgl-miR160a-3p was consistent with the sequencing result. This study clarifies the molecular mechanism of R. glutinosa in response to GG22 stress, laying a theoretical basis for the improvement and future research of R. glutinosa.

Label-free photoacoustic computed tomography of mouse cortical responses to retinal photostimulation using a pair-wise correlation map.

The lack of a non-invasive or minimally invasive imaging technique has long been a challenge to investigating brain activities in mice. Functional magnetic resonance imaging and the more recently developed diffuse optical imaging both suffer from limited spatial resolution. Photoacoustic (PA) imaging combines the sensitivity of optical excitation to hemodynamic changes and ultrasound detection's relatively high spatial resolution. In this study, we evaluated the feasibility of using a label-free, real-time PA computed tomography (PACT) system to measure visually evoked hemodynamic responses within the primary visual cortex (V1) in mice. Photostimulation of the retinas evoked significantly faster and stronger V1 responses in wild-type mice than in age-matched rod/cone-degenerate mice, consistent with known differences between rod/cone- vs. melanopsin-mediated photoreception. In conclusion, the PACT system in this study has sufficient sensitivity and spatial resolution to resolve visual cortical hemodynamics during retinal photostimulation, and PACT is a potential tool for investigating visually evoked brain activities in mouse models of retinal diseases.

Photoacoustic imaging of squirrel monkey cortical and subcortical brain regions during peripheral electrical stimulation.

The investigation of neuronal activity in non-human primate models is of critical importance due to their genetic similarity to human brains. In this study, we tested the feasibility of using photoacoustic imaging for the detection of cortical and subcortical responses due to peripheral electrical stimulation in a squirrel monkey model. Photoacoustic computed tomography and photoacoustic microscopy were applied on squirrel monkeys for real-time deep subcortical imaging and optical-resolution cortical imaging, respectively. The electrically evoked hemodynamic changes in primary somatosensory cortex, premotor cortices, primary motor cortex, and underlying subcortical areas were measured. Hemodynamic responses were observed in both cortical and subcortical brain areas at the cortices during external stimulation, demonstrating the feasibility of photoacoustic technique for functional imaging of non-human primate brain.

Hybrid photonic bandgap effect in twisted hollow-core photonic bandgap fibers.

A hybrid photonic bandgap effect in twisted hollow-core photonic bandgap fibers (HC-PBFs) is theoretically investigated for the first time, to the best of our knowledge. Due to the topological effect, twisting of the fibers changes the effective refractive index and lifts the degeneracy of the photonic bandgap ranges of the cladding layers. This twist-induced hybrid photonic bandgap effect shifts up the center wavelength and narrows the bandwidth of the transmission spectrum. A quasi-single-mode low-loss transmission is achieved in the twisted 7-cell HC-PBFs with a twisting rate α = 7-8 rad/mm, which has a loss < 30 dB/km and higher-order mode extinction ratio > 15 dB. The twisted HC-PBFs could be suitable for applications such as spectral and mode filters.

The feasibility study of the transmission mode photoacoustic measurement of human calcaneus bone in vivo.

The photoacoustic (PA) technique is uniquely positioned for biomedical applications primarily due to its ability to visualize optical absorption contrast in deep tissue at ultrasound resolution. In this work, via both three-dimensional (3D) numerical simulations and in vivo experiments on human subjects, we investigated the possibility of PA measurement of human calcaneus bones in vivo in a non-invasive manner, as well as its feasibility to differentiate osteoporosis patients from normal subjects. The results from the simulations and the experiments both demonstrated that, when one side of the heel is illuminated by laser with light fluence under the ANSI safety limit, the PA signal generated in the human calcaneus bone can be detected by an ultrasonic transducer at the other side of the heel (i.e. transmission mode). Quantitative power spectral analyses of the calcaneus bone PA signals were also conducted, demonstrating that the microarchitectural changes in calcaneus bone due to osteoporosis can be detected, as reflected by enhanced high frequency components in detected PA bone signal. Further statistical analysis of the experimental results from 10 osteoporosis patients and 10 healthy volunteers showed that the weighted frequency as a quantified PA spectral parameter can differentiate the two subject groups with statistical significance.

Single-polarization single-mode hollow-core photonic-bandgap fiber with thin slab waveguide.

A novel single-polarization single-mode hollow-core photonic bandgap fiber with thin slab waveguide (TSW) was designed and simulated. Single-polarization guidance is achieved by the high loss of a polarized fundamental mode (FM) induced by mode coupling with a higher-order TE/TM mode of TSW and low loss of another polarized FM. We achieve a polarization loss ratio ∼ 46.9 dB, birefringence Δn ∼ 2.4 × 10-4, loss ∼ 35.9 dB/km and minimum higher-order mode extinction ratio > 15 dB. Moreover, the performance could be maintained when the guidance wavelength λ = 1.44 ∼ 1.56 µm and bending radius rc > 9 mm. The proposed model will be suitable for application as resonator sensing paths of miniaturized resonator fiber optic gyroscopes, high-performance interferometers, fiber lasers, frequency metrology, quantum communications, and laser-based gas sensing, etc.

Comparative transcriptome analysis of the hyperaccumulator plant Phytolacca americana in response to cadmium stress.

To study the molecular mechanism of the hyperaccumulator plant Phytolacca americana against cadmium (Cd) stress, the leaves of P. americana treated with 400 µM Cd for 0, 2, 12, and 24 h were harvested for comparative transcriptome analysis. In total, 110.07 Gb of clean data were obtained, and 63,957 unigenes were acquired after being assembled. Due to the lack of P. americana genome information, only 24,517 unigenes were annotated by public databases. After Cd treatment, 5054 differentially expressed genes (DEGs) were identified. KEGG pathway enrichment analysis of DEGs showed that genes involved in the flavonoid biosynthesis and antenna proteins of photosynthesis were significantly down-regulated, while genes related to the lignin biosynthesis pathway were remarkably up-regulated, indicating that P. americana could synthesize more lignin to cope with Cd stress. Moreover, genes related to heavy metal accumulation, sulfur metabolism and glutathione metabolism were also significantly up-regulated. The gene expression pattern of several key genes related to distinct metabolic pathways was verified by qRT-PCR. The results indicated that the immobilization of lignin in cell wall, chelation, vacuolar compartmentalization, as well as the increase of thiol compounds content may be the important mechanisms of Cd detoxification in hyperaccumulator plant P. americana. Accession numbers: the raw data of P. americana transcriptome presented in this study are openly available in NCBI SRA database, under the BioProject of PRJNA649785. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02865-x.

Primary intraosseous malignancies: A 10-year retrospective cohort study.

OBJECTIVE: This study aimed to explore the characteristics and prognoses of primary jaw malignancies and identify valuable prognostic factors. STUDY DESIGN: Patients who were primarily diagnosed with jaw malignancies at Beijing Stomatological Hospital between 2006 and 2016 were retrospectively reviewed. RESULTS: Eighty patients with malignant jaw tumors were enrolled in the study. Squamous cell carcinoma was the most common type (56.2%). The age range of patients varied from 11 to 92 years old (mean age = 51.5 years), and the male-to-female ratio was 2.1:1. Anatomically, the mandible was involved in 82.5% of cases. The estimated disease-free survival and overall survival (OS) rates were 60.6% and 73.2%, respectively. Cox multivariate analysis showed that a smoking history (hazard ratio = 5.015, 95% confidence interval, 1.900-13.235, P = .001) and advanced age (≥60 years; hazard ratio = 2.464, 95% confidence interval, 0.987-6.155, P = .046) were independent prognostic factors for poor OS. CONCLUSION: Primary jaw malignancies were more prevalent in the mandible and among males and middle-aged patients. Radical resection of the primary surgery was the basis of a good prognosis, and the choice of postoperative adjuvant therapy may not ultimately affect OS. Patients with a history of smoking and advanced age were at high risk for poor prognosis.