Spinal cord and brain atrophy patterns in neuromyelitis optica spectrum disorder and multiple sclerosis.

BACKGROUND: Spinal cord and brain atrophy are common in neuromyelitis optica spectrum disorder (NMOSD) and relapsing-remitting multiple sclerosis (RRMS) but harbor distinct patterns accounting for disability and cognitive impairment. METHODS: This study included 209 NMOSD and 304 RRMS patients and 436 healthy controls. Non-negative matrix factorization was used to parse differences in spinal cord and brain atrophy at subject level into distinct patterns based on structural MRI. The weights of patterns were obtained using a linear regression model and associated with Expanded Disability Status Scale (EDSS) and cognitive scores. Additionally, patients were divided into cognitive impairment (CI) and cognitive preservation (CP) groups. RESULTS: Three patterns were observed in NMOSD: (1) Spinal Cord-Deep Grey Matter (SC-DGM) pattern was associated with high EDSS scores and decline of visuospatial memory function; (2) Frontal-Temporal pattern was associated with decline of language learning function; and (3) Cerebellum-Brainstem pattern had no observed association. Patients with CI had higher weights of SC-DGM pattern than CP group. Three patterns were observed in RRMS: (1) DGM pattern was associated with high EDSS scores, decreased information processing speed, and decreased language learning and visuospatial memory functions; (2) Frontal-Temporal pattern was associated with overall cognitive decline; and (3) Occipital pattern had no observed association. Patients with CI trended to have higher weights of DGM and Frontal-Temporal patterns than CP group. CONCLUSION: This study estimated the heterogeneity of spinal cord and brain atrophy patterns in NMOSD and RRMS patients at individual level, and evaluated the clinical relevance of these patterns, which may contribute to stratifying participants for targeted therapy.

A radiomics-based brain network in T1 images: construction, attributes, and applications.

T1 image is a widely collected imaging sequence in various neuroimaging datasets, but it is rarely used to construct an individual-level brain network. In this study, a novel individualized radiomics-based structural similarity network was proposed from T1 images. In detail, it used voxel-based morphometry to obtain the preprocessed gray matter images, and radiomic features were then extracted on each region of interest in Brainnetome atlas, and an individualized radiomics-based structural similarity network was finally built using the correlational values of radiomic features between any pair of regions of interest. After that, the network characteristics of individualized radiomics-based structural similarity network were assessed, including graph theory attributes, test-retest reliability, and individual identification ability (fingerprinting). At last, two representative applications for individualized radiomics-based structural similarity network, namely mild cognitive impairment subtype discrimination and fluid intelligence prediction, were exemplified and compared with some other networks on large open-source datasets. The results revealed that the individualized radiomics-based structural similarity network displays remarkable network characteristics and exhibits advantageous performances in mild cognitive impairment subtype discrimination and fluid intelligence prediction. In summary, the individualized radiomics-based structural similarity network provides a distinctive, reliable, and informative individualized structural brain network, which can be combined with other networks such as resting-state functional connectivity for various phenotypic and clinical applications.

A Frontline, Rapid, Nucleic Acid-Based Fusarium circinatum Detection System Using CRISPR/Cas12a Combined with Recombinase Polymerase Amplification.

Pitch canker caused by the fungus Fusarium circinatum is a damaging disease that affects pines in Europe, South Africa, and North America in both the southeast and west coast of the United States. Several countries, including China, have listed F. circinatum as a quarantine pathogen. Timely detection, an important pillar of the quarantine effort, can efficiently prevent the introduction of F. circinatum into new areas or facilitate management and eradication strategies in already infested sites. In this study, we developed an F. circinatum detection technique based on a combination of recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology (termed RPA-CRISPR/Cas12a). After obtaining DNA, this novel method can be utilized for the molecular identification of F. circinatum using the naked eye and can specifically detect F. circinatum at DNA concentrations as low as 200 fg within 30 min at 37°C. The system is sensitive for both standard laboratory samples and samples from the field. In summary, we have developed a simple, rapid, sensitive, unaided-eye visualization, RPA-CRISPR/Cas12a-based detection system for the molecular identification of F. circinatum that does not require technical expertise or expensive ancillary equipment.

Recombinase Polymerase Amplification-Lateral Flow Dipstick Assay for Rapid Detection of Fusarium circinatum Based on a Newly Identified Unique Target Gene.

Pitch canker caused by the fungus Fusarium circinatum is an important disease affecting pine trees in Europe and South Africa. Several countries, including China, have listed F. circinatum as a quarantine pathogen. Therefore, timely detection of F. circinatum could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay was developed for rapid detection of F. circinatum based on a new target gene, Fcir2067, identified from whole-genome sequences. The assay was highly specific to F. circinatum. In fact, it exclusively detected F. circinatum isolates; 53 isolates of fungal and oomycete species and 2 nematodes of Bursaphelenchus xylophilus and B. mucronatus were not detected. By detecting as little as 10 pg of F. circinatum genomic DNA in a 50-µl reaction, the RPA-LFD assay was 10 times more sensitive than conventional PCR assays. F. circinatum was also detected in artificially inoculated pine needles of Cedrus deodara. These results demonstrated that the developed RPA-LFD assay has the potential for rapid detection of F. circinatum in regions at high risk of infection. The RPA-LFD assay might serve as an alternative method for the early detection of F. circinatum.

Rapid detection of Phytophthora cinnamomi based on a new target gene Pcinn13739.

Phytophthora cinnamomi causes crown and root wilting in more than 5,000 plant species and represents a significant threat to the health of natural ecosystems and horticultural crops. The early and accurate detection of P. cinnamomi is a fundamental step in disease prevention and appropriate management. In this study, based on public genomic sequence data and bioinformatic analysis of several Phytophthora, Phytopythium, and Pythium species, we have identified a new target gene, Pcinn13739; this allowed us to establish a recombinase polymerase amplification-lateral flow dipstick (RPA-LFD) assay for the detection of P. cinnamomi. Pcinn13739-RPA-LFD assay was highly specific to P. cinnamomi. Test results for 12 isolates of P. cinnamomi were positive, but negative for 50 isolates of 25 kinds of Phytophthora species, 13 isolates of 10 kinds of Phytopythium and Pythium species, 32 isolates of 26 kinds of fungi species, and 11 isolates of two kinds of Bursaphelenchus species. By detecting as little as 10 pg.µl-1 of genomic DNA from P. cinnamomi in a 50-µl reaction, the RPA-LFD assay was 100 times more sensitive than conventional PCR assays. By using RPA-LFD assay, P. cinnamomi was also detected on artificially inoculated fruit from Malus pumila, the leaves of Rhododendron pulchrum, the roots of sterile Lupinus polyphyllus, and the artificially inoculated soil. Results in this study indicated that this sensitive, specific, and rapid RPA-LFD assay has potentially significant applications to diagnosing P. cinnamomi, especially under time- and resource-limited conditions.

A Reparametrized CNN Model to Distinguish Alzheimer's Disease Applying Multiple Morphological Metrics and Deep Semantic Features From Structural MRI.

It is of potential clinical value to improve the accuracy of Alzheimer's disease (AD) recognition using structural MRI. We proposed a reparametrized convolutional neural network (Re-CNN) to discriminate AD from NC by applying morphological metrics and deep semantic features. The deep semantic features were extracted through Re-CNN on structural MRI. Considering the high redundancy in deep semantic features, we constrained the similarity of the features and retained the most distinguishing features utilizing the reparametrized module. The Re-CNN model was trained in an end-to-end manner on structural MRI from the ADNI dataset and tested on structural MRI from the AIBL dataset. Our proposed model achieves better performance over some existing structural MRI-based AD recognition models. The experimental results show that morphological metrics along with the constrained deep semantic features can relatively improve AD recognition performance. Our code is available at: https://github.com/czp19940707/Re-CNN.

An Integrated Model Combining Machine Learning and Deep Learning Algorithms for Classification of Rupture Status of IAs.

Background: The rupture risk assessment of intracranial aneurysms (IAs) is clinically relevant. How to accurately assess the rupture risk of IAs remains a challenge in clinical decision-making. Purpose: We aim to build an integrated model to improve the assessment of the rupture risk of IAs. Materials and Methods: A total of 148 (39 ruptured and 109 unruptured) IA subjects were retrospectively computed with computational fluid dynamics (CFDs), and the integrated models were proposed by combining machine learning (ML) and deep learning (DL) algorithms. ML algorithms that include random forest (RF), k-nearest neighbor (KNN), XGBoost (XGB), support vector machine (SVM), and LightGBM were, respectively, adopted to classify ruptured and unruptured IAs. A Pointnet DL algorithm was applied to extract hemodynamic cloud features from the hemodynamic clouds obtained from CFD. Morphological variables and hemodynamic parameters along with the extracted hemodynamic cloud features were acted as the inputs to the classification models. The classification results with and without hemodynamic cloud features are computed and compared. Results: Without consideration of hemodynamic cloud features, the classification accuracy of RF, KNN, XGB, SVM, and LightGBM was 0.824, 0.759, 0.839, 0.860, and 0.829, respectively, and the AUCs of them were 0.897, 0.584, 0.892, 0.925, and 0.890, respectively. With the consideration of hemodynamic cloud features, the accuracy successively increased to 0.908, 0.873, 0.900, 0.926, and 0.917. Meanwhile, the AUCs reached 0.952, 0.881, 0.950, 0.969, and 0.965 eventually. Adding consideration of hemodynamic cloud features, the SVM could perform best with the highest accuracy of 0.926 and AUC of 0.969, respectively. Conclusion: The integrated model combining ML and DL algorithms could improve the classification of IAs. Adding consideration of hemodynamic cloud features could bring more accurate classification, and hemodynamic cloud features were important for the discrimination of ruptured IAs.

First Report of Crown and Root Rot Caused by Fusarium oxysporum on Photinia × fraseri Dress in China.

Photinia × fraseri Dress was introduced to China in 1998 and has been widely used in gardens as an ornamental plant. From April 2021 to 2022, a diseasecausing blight and root discoloration in approximately 80% of P. × fraseri at several landscape sites in Xuanwu Lake Park. Symptomatic root tissues were immersed in 75% ethanol for 30 s followed by 1% NaClO for 90 s, rinsed with sterile water 3 times, and placed on Potato Dextrose Agar (PDA). After 3 days of dark incubation at 25 °C, white Fusarium-like colonies grew out from the symptomatic root tissue pieces. Three representative isolates (SG1, SG6, and SG23) were obtained and deposited in China's Forestry Culture Collection Center. The hyphae grew radially and the aerial hyphae were velvety, white or pinkish-white . After 20 days, macroconidia, microconidia, and chlamydospores were produced in the colonies on PDA. Macroconidia are sickle-shaped, slightly curved, 23-50.6 µm × 4-6 µm in size. Microconidia were numerous, oval or kidney-shaped, 6.7-12.6 µm × 3.5-5.7 µm in size, with germinating from one or both ends (Fig S1E, F). Chlamydospores were spherical, smooth, and round, in chains or solitary in hyphae (Fig S1G). All three isolates had identical morphological features. Phylogenetic analysis of concatenated CAMD, RPB2 and TEF1 sequences showed that the three isolates clustered in the same clade as F. oxysporum. Two-year old P. × fraseri potted seedlings (30-cm tall, n=12) were placed at the greenhouse (temperature; 26°C, daylight; 14 hours) for the pathogenicity tests . Roots of P. × fraseri were dipped in a 10 mL of the conidial suspension (106 conidia/mL) of each isolate for 2 hours, and the control plants were inoculated with sterile water. Results showed that after 21 days post-inoculation, all inoculated seedlings (n=9) showed crown and root rot . In contrast, none of the control seedlings (n=3) were affected. Re-isolation of three fungal isolates (infected root) showed that their morphology and gene markers sequence were identical to the original isolates thus fulfilled Koch's postulates. Globally, this is the first report of F. oxysporum causing crown blight and root rot of P. × fraseri, which is also a potential threat to the two parent hosts (P. serratifolia and P. glabra). Additional surveys are being conducted for mapping the distribution of F. oxysporum in the Nanjing Province of China.

Emojis predict dropouts of remote workers: An empirical study of emoji usage on GitHub.

Emotions at work have long been identified as critical signals of work motivations, status, and attitudes, and as predictors of various work-related outcomes. When more and more employees work remotely, these emotional signals of workers become harder to observe through daily, face-to-face communications. The use of online platforms to communicate and collaborate at work provides an alternative channel to monitor the emotions of workers. This paper studies how emojis, as non-verbal cues in online communications, can be used for such purposes and how the emotional signals in emoji usage can be used to predict future behavior of workers. In particular, we present how the developers on GitHub use emojis in their work-related activities. We show that developers have diverse patterns of emoji usage, which can be related to their working status including activity levels, types of work, types of communications, time management, and other behavioral patterns. Developers who use emojis in their posts are significantly less likely to dropout from the online work platform. Surprisingly, solely using emoji usage as features, standard machine learning models can predict future dropouts of developers at a satisfactory accuracy. Features related to the general use and the emotions of emojis appear to be important factors, while they do not rule out paths through other purposes of emoji use.

A Dissipation Function-Based Method for Calculating the Energy Loss of Intracranial Aneurysms.

At present, the energy loss (EL) mechanism of intracranial aneurysm (IA) rupture is explored based on the global EL calculated by Bernoulli equation, but the details of EL are still unclear. This study aimed to explore the temporal and spatial characteristics of EL of IAs and reveal its mechanism. A novel method for calculating the EL of IAs based on dissipation function (DF) was proposed. DF was derived from the differential form of the energy equation and reflected the irreversible conversion from mechanical energy to internal energy caused by the friction between the fluid micelles. Eight sidewall IAs located at the posterior communicating segment of the internal carotid artery were collected; the three-dimensional (3D) geometric models of IAs were established employing image segmentation and 3D reconstruction. Computational fluid dynamics was applied to obtain hemodynamic parameters of IAs. The temporal and spatial characteristics of EL of IAs were achieved utilizing our proposed method. The simulation results indicated that EL occurred mainly in the boundary layer and the region adjacent to high-velocity inflow jet, EL increased rapidly during cardiac systole and reached its maximum at end-systolic phase and then decreased gradually during diastole until the end of cardiac cycle. The proposed method achieved some improvements over the traditional Bernoulli equation-based method by acquiring the temporal and spatial characteristics of EL, and it could provide insights into the EL of IAs and contribute to further rupture mechanism investigation.

Evaluation of characteristics and microbial community of anaerobic granular sludge under microplastics and aromatic carboxylic acids exposure.

The influences of polyether sulfone (PES) microplastics and different structures aromatic carboxylic acids such as benzoic acid (BA), phthalic acid (PA), hemimellitic acid (HA), and 1-naphthoic acid (1-NA) on the performances and characteristics of anaerobic granular sludge as well as the microbial community were investigated. The chemical oxygen demand (COD) removal efficiency was the highest in the experimental group with 40 mg/L BA, reaching 90.1%. The inhibitory effect of aromatic carboxylic acids addition on the 2,3,5-triphenyltetrazolium chloride (TTC) activity was more obvious than that on 2-para (iodo-phenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) activity. Compared with the control group (only 0.5 g/L PES microplastics, 60.6 mg TF·g TSS·h-1), the inhibition effect of TTC activity was 32.5 mg TF·g TSS·h-1 and 44.3 mg TF·g TSS·h-1 in the 40 mg/L HA and 40 mg/L 1-NA experimental groups, respectively. When aromatic carboxylic acids were added, the activities of acetate kinase and coenzyme F420 in the anaerobic granular sludge decreased. The excitation-emission matrix (EEM) fluorescence spectra indicated that loosely bound extracellular polymeric substances (LB-EPS) began to decay. After the addition of different aromatic carboxylic acids, the CC and CH functional groups of the anaerobic granular sludge increased, suggesting that aromatic carboxylic acids migrated to the surface of anaerobic granular sludge, such a transfer would lead to changes in anaerobic granular sludge performance. High-throughput sequencing technology showed that the dominant microbial communities in the anaerobic granular sludge were Proteobacteria, Methanothrix, and Methanomicrobia. After the addition of aromatic carboxylic acids, the relative abundances of Proteobacteria, Methanobacterium, and Methanospirillum increased. In the presence of PES, 1-NA had the most serious toxicity to the anaerobic granular sludge.

First Report of Phytophthora pini Causing Foliage Blight and Shoot Dieback of Rhododendron pulchrum in China.

During a 2019-2020 survey for plant pathogenic oomycetes in Nanjing, China, severe foliage blight and dieback were observed on approximately 20 Rhododendron pulchrum plants at three public parks and gardens. Approximately 25% of leaves and shoots were affected. Symptoms included brown to black lesions on leaves and stems, dieback of shoot tips, and wilting. Diseased tissues were collected from a five-year-old shrub with typical disease symptoms at Xuanwuhu Park. They were cut into 10×10 mm2 squares, immersed in 70% ethanol for 30 sec, and placed onto fresh clarified V8 juice agar (cV8A) containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene. Phytophthora-like hypae were transferred to new cV8A plates daily. A total of five isolates were obtained after five days of incubation at 25°C. After approximately 20 days, all isolates were identical in morphological traits including semi-papillate sporangia and gametangia (homothallic). Thirty sporangia of a representative isolate Ppi were randomly selected and examined. They were mostly ovoid and sometimes obpyriform, averaging 41.0 ± 3.9 × 24.8 ± 3.2 µm. Antheridia of 30 randomly selected gametangia were paragynous, averaging 16.7 ± 0.7 × 12.4 ± 1.5 µm. Average diameters of oogonia and plerotic oospores were 29.2 ± 0.3 µm and 26.4 ± 1.6 µm, respectively. Chlamydospores were not observed. The above morphological traits suggested the causal agent belonging to the "P. citricola-complex". Isolate Ppi was subjected to sequencing of the rDNA internal transcribed spacer (ITS) region and the ras-related GTP-binding protein 1 (Ypt1) gene. ITS sequence of Ppi (GenBank ACN. MT672594) has 100% identity to that of P. pini (MG865565). It has a 3-nt difference from the ITS sequences of P. acerina (MG518642) and P. citricola (MG865475) and a 4-nt difference from that of P. plurivora (FJ665225). Ypt1 sequence of Ppi (MT680000) has 100% identity to that of P. pini (MK058416). Pathogenicity of Ppi on R. pulchrum was tested using both detached-leaf and whole-plant assays. In the former assay, each of six asymptomatic leaves was symmetrically wounded at both sides using a sterile inoculation needle. A 5×5 mm2 Ppi-colonized cV8A plug was placed on each wound of five leaves. Sterile agar plugs were used for a control leaf. All six leaves were placed on a wet filter paper in a closed container at 25°C. This assay was repeated twice. On the fifth day, all inoculated leaves had necrotic tissues around the wounds, while the control leaves remained asymptomatic. In the whole-plant assay, 20-inch-tall plants were used. Five attached leaves and the twig base of each plant were wounded. A control plant was inoculated in the same manner above, while sterile agar plugs were used. Each plant was covered with a plastic bag and maintained at 25°C. Wet cotton balls were placed in the bags to maintain humidity. After two days, the bag containing cotton balls was removed. This assay was repeated three times. After two weeks, all three inoculated plants in the three replicated trials had severe foliage blight and dieback, whereas control plants remained healthy. Phytophthora isolates recovered from artificially inoculated tissues were identical to isolate Ppi in morphological characters. Rhododendron diseases caused by P. pini were reported in the USA and Finland . This is the first report of P. pini causing foliage blight and dieback on R. pulchrum, an important nursery and landscape plant in China. Additional surveys are ongoing to determine the distribution of this pathogen in Nanjing. Management programs are under development to contain the spread of P. pini and treat diseased plants.

First Report of Phytopythium helicoides Causing Crown and Root Rot on Rhododendron pulchrum in China.

During a 2019-2020 survey of plant pathogenic oomycetes in Nanjing, China, a cluster of five adjacent Rhododendron pulchrum plants in Xuanwuhu Park exhibited symptoms including crown and root rot and wilting. foliage blight caused due to collar and had rotting crown and root tissues resultingrot foliage blight. Diseased roots were rinsed in water, cut into 10 mm pieces, immersed in 70% ethanol for 60 sec, and plated onto clarified V8 juice agar (cV8A) containingamended with pimaricin (20 mg/liter), ampicillin (125 mg/liter), rifampicin (10 mg/liter), and pentachloronitrobenzene (20 mg/liter). After three3 days of incubation at 26°C, Ffive Pythium-like isolatescoloniesisolates were obtained using hypalhyphal-tipping after 3 days of incubation at 25°C. Ten agar plugs (2×2 mm2) of each isolate were growntransferred into 10 mLl of 10% clarified V8 juice (cV8) in a 100 -mm plate at 26°C to produce mycelial mats. After 3three days, cV8 was replaced with sterile water. To stimulate sporangial production, 3-5 drops of soil extract solution were added to each plate. Five isolates had identical morphological features. Sporangia were terminal, ovoid to globose, andmeasuring 34.2 ± 6.2 µm (24.0-42.5 µm range) in length and 30.7 ± 6.6 µm (20.9-41.1 µm range) in width. Oogonia were not observed. The following primers were used to amplify the rDNA internal transcribed spacer (ITS) region and the mitochondrial cytochrome c oxidase subunit 1 (cox1COI) and 2 (cox2COII) genes  of from aA representative isolate, PH-C were amplified using the primer pairs ITS6 and ITS4 (Cooke et al. 2000), OomCoxI-Levup and OomCoxI-Levlo (Robideau et al. 2011) and Cox2-F and Cox2-RC4 (Hudspeth et al. 2000), respectivelyPhe-1. Isolate A xxx675 bp, xxx657 bp and 561xxx bp fragmentPH-C , respectively were amplified and had have identical sequences of the ITS (GenBank ACN. MT824568), and cox1 (MT834959), COI and cox2 COII genes the rDNA internal transcribed spacer (ITS) region and the mitochondrial cytochrome c oxidase subunit 1 and 2 genes (GenBank ACN. MT824568, MT834959, (MT834958, respectively) sequences identical to those of Phytopythium helicoides (MN541109, MK879709, KT595689, respectively). Based on the morphological and molecular characters, all five isolatesthe causal agent waswere identified the species represented by Phe-1 was identified as P. helicoides. One-year-old R. pulchrum plants (approx. 0.3 m in height) grown in 8×8 cm2 pots were used in to test the pathogenicity trials. Ten plants wasere carefully dug up to expose root ballsclusterballs. TenThree- days -old cultures of the isolate PH-Che-1 were used as the inoculum. Five The pplantss wereere inoculated by inserting 10 agar plugs into thee root ball of each plantcluster. For inoculatingfive control plants, sterile cV8A discsplugs were used. All inoculated plants were re-potted using original fresh potting mix and potsture .Ten 3-day-old cV8A cultural plugs (5×5 mm2) of Phe-1 were evenly insert into the root ball of each of five plants, while sterile cV8A plugs were used for five control plants. All were then planted into their original pots. Plants were maintained in a growth chamber set at 26°C with a 12/12 h light/dark cycle and irrigated as needed. After 21-25 days, the inoculated plants had symptoms identical to those in the field, while the controls remained asymptomatic. Identical outcomes were obtained from two repeated The pathogenicity trials. test was repeatedconducted twice . and the coutcome was identical. Phytopythium. helicoides (Phe-1) was reisolated from all symptomatic plants inemerging from the pathogenicity trials. Phytopythium helicoides was found causing diseases of Asian lotus (Yin et al. 2015), mandarin orange (Chen et al. 2016), and kiwifruit (Wang et al. 2015) plants in China. Phytopythium isolates with identical morphological features to those of Phe-1 were recovered from rotted crown and root tissues of all inoculated plants. In this note, P. helicoides causing crown and root rot on R. pulchrum is reported for the first time. Globally, this is the first report of P. helicoides causing crown blight and root rot of R. pulchrum. Additional surveys are being conducted forto mapping the distribution of P. helicoides in Nanjing, Province of China.

The application of the scallop nanostructure in deep silicon etching.

Micro/nanostructures with high aspect ratios in silicon wafers obtained by plasma etching are of great significance in device fabrication. In most cases, the scallop nanostructure in deep silicon etching should be suppressed. However, the scallop nanostructure could be applied in electronic device fabrication as characteristic information, which indicates the balance between deposition and etching. In this work, the applications of scallop nanostructures in etching process optimization and environmental protection are demonstrated. In addition, the minimum effect of the cycle time on the scallop size is reported for the first time. These results could bring new thoughts to the electronic devices related fields, such as micro-electro-mechanical systems (MEMS), silicon capacitors and advanced packaging.