Coupled hydraulics and carbon economy underlie age-related growth decline and revitalisation of sand-fixing shrubs after crown removal.

Crown removal revitalises sand-fixing shrubs that show declining vigour with age in drought-prone environments; however, the underlying mechanisms are poorly understood. Here, we addressed this knowledge gap by comparing the growth performance, xylem hydraulics and plant carbon economy across different plant ages (10, 21 and 33 years) and treatments (control and crown removal) using a representative sand-fixing shrub (Caragana microphylla Lam.) in northern China. We found that growth decline with plant age was accompanied by simultaneous decreases in soil moisture, plant hydraulic efficiency and photosynthetic capacity, suggesting that these interconnected changes in plant water relations and carbon economy were responsible for this decline. Following crown removal, quick resprouting, involving remobilisation of root nonstructural carbohydrate reserves, contributed to the reconstruction of an efficient hydraulic system and improved plant carbon status, but this became less effective in older shrubs. These age-dependent effects of carbon economy and hydraulics on plant growth vigour provide a mechanistic explanation for the age-related decline and revitalisation of sand-fixing shrubs. This understanding is crucial for the development of suitable management strategies for shrub plantations constructed with species having the resprouting ability and contributes to the sustainability of ecological restoration projects in water-limited sandy lands.

Experimental recognition of vortex beams in oceanic turbulence combining the Gerchberg-Saxton algorithm and convolutional neural network.

In underwater wireless optical communication (UWOC), vortex beams carrying orbital angular momentum (OAM) can improve channel capacity but are vulnerable to oceanic turbulence (OT), leading to recognition errors. To mitigate this issue, we propose what we believe to be a novel method that combines the Gerchberg-Saxton (GS) algorithm-based recovery with convolutional neural network (CNN)-based recognition (GS-CNN). Our experimental results demonstrate that superposed Laguerre-Gaussian (LG) beams with small topological charge are ideal information carriers, and the GS-CNN remains effective even when OT strength C n2 is high up to 10-11 K 2 m -2/3. Furthermore, we use 16 kinds of LG beams to transmit a 256-grayscale digital image, giving rise to an increase in recognition accuracy from 0.75 to 0.93 and a decrease in bit error ratio from 3.98×10-2 to 6.52×10-3 compared to using the CNN alone.

Characterization and phylogenetic analysis of a neurovirulent Zika virus isolated from Cambodia in 2019.

The geographic range of Zika virus (ZIKV) has expanded from Asia to the Americas, leading to the 2015-2016 pandemic with enhanced neurovirulence. At present, ZIKV is continuously circulating in many Southeast Asian countries. Unfortunately, the persistent evolution of ZIKV in Southeast Asia and its influence on the biological characteristics of the virus remain incompletely understood. In this study, the in vitro and in vivo properties of a new ZIKV isolate obtained from Cambodia in 2019 (CAM/2019) were characterized and compared with those of the Cambodian strain (CAM/2010). Compared with CAM/2010, the CAM/2019 virus showed similar plaque morphology and growth curves in cell cultures and induced comparable viremia and organ viral loads profiles in both BALB/c and A129 (IFNAR1-/- ) mice upon intraperitoneal (i.p.) inoculation. Remarkably, the CAM/2019 virus exhibited enhanced neurovirulence in neonatal mice compared with CAM/2010, with a 74-fold reduction in the 50% lethal dose (LD50 ). Consistently, CAM/2019 produced higher viral loads in the brains of BALB/c neonatal mice than CAM/2010 did. Sequence alignment showed that the CAM/2019 virus has acquired 12 amino acid substitutions, several of which were found to be associated with neurovirulence. In particular, the CAM/2019 virus shared an A1204T substitution in NS2A with the Thai isolate SI-BKK02 that was isolated from a microcephaly case. Taken together, our results indicate that a ZIKV strain isolated with specific mutations has emerged in Cambodia, highlighting the need for extensive molecular and disease surveillance in Cambodia and other Asian countries.

Novel Isomer of Volleyballene Sc20C60.

The Stone-Wales defect is a well-known and significant defective structure in carbon materials, impacting their mechanical, chemical, and electronic properties. Recently, a novel metal-carbon nanomaterial named Volleyballene was discovered, characterized by a C-C bond bridging two carbon pentagons. Using first-principles calculations, a stable Stone-Wales-defective counterpart of Volleyballene, exhibiting Th symmetry, has been proposed by rotating the C-C bond by 90°. Although its binding energy per atom is slightly higher than that of Volleyballene (ΔEb = 0.009 eV/atom), implying marginally lower structural stability, it can maintain its bond structure until the effective temperature reaches about 1500 K, indicating greater thermodynamic stability. Additionally, its highest vibration frequency is 1346.2 cm-1, indicating a strong chemical bond strength. A theoretical analysis of the Sc20C60 + Sc20C60 binary systems highlights that the stable building block may be applied in potential nanoassemblies.

Application of ultrasonography in predicting the biological risk of gastrointestinal stromal tumors.

OBJECTIVES: To explore and establish a reliable and noninvasive ultrasound model for predicting the biological risk of gastrointestinal stromal tumors (GISTs). MATERIALS AND METHODS: We retrospectively reviewed 266 patients with pathologically-confirmed GISTs and 191 patients were included. Data on patient sex, age, tumor location, biological risk classification, internal echo, echo homogeneity, boundary, shape, blood flow signals, presence of necrotic cystic degeneration, long diameter, and short/long (S/L) diameter ratio were collected. All patients were divided into low-, moderate-, and high-risk groups according to the modified NIH classification criteria. All indicators were analyzed by univariate analysis. The indicators with inter-group differences were used to establish regression and decision tree models to predict the biological risk of GISTs. RESULTS: There were statistically significant differences in long diameter, S/L ratio, internal echo level, echo homogeneity, boundary, shape, necrotic cystic degeneration, and blood flow signals among the low-, moderate-, and high-risk groups (all p < .05). The logistic regression model based on the echo homogeneity, shape, necrotic cystic degeneration and blood flow signals had an accuracy rate of 76.96% for predicting the biological risk, which was higher than the 72.77% of the decision tree model (based on the long diameter, the location of tumor origin, echo homogeneity, shape, and internal echo) (p = .008). In the low-risk and high-risk groups, the predicting accuracy rates of the regression model reached 87.34 and 81.82%, respectively. CONCLUSIONS: Transabdominal ultrasound is highly valuable in predicting the biological risk of GISTs. The logistic regression model has greater predictive value than the decision tree model.

Evaluation of macular microvascular density using optical coherence tomography angiography in patients with Posner-Schlossman syndrome.

BACKGROUND: Optical coherence tomography angiography (OCTA) is a novel technology that provides a noninvasive, dye-less method to visualize the blood vessels of the retina. In the present study, we investigate macular microvascular density and the correlation of ocular and demographic factors using OCTA in Posner-Schlossman syndrome (PSS) patients. METHODS: This is a prospective observational study. All PSS patients and age- and sex-matched healthy subjects underwent complete ophthalmologic examination, and RE, BCVA, IOP, CCT, AL, CMT, GCIPI, RNFL, C/D ratio were recorded. The whole-image vessel density (wiVD) and whole-image perfusion density (wiPD), three-circle (1 mm central ring, 3 mm inner ring, 6 mm outer ring), and four-quadrant segmental VD and PD were calculated. RESULTS: Seventeen PSS patients and 17 healthy subjects were enrolled in this study. The mean age was 42.65 ± 11.22 years in PSS patients and 42.71 ± 10.50 years in healthy controls. IOP, CCT, and C/D ratio were higher in PSS-attacked eyes, and BCVA, OPP and RNFL thickness was lower than those in the fellow eyes (p < 0.05). BCVA and OPP were improved in the PSS-attacked eyes in intermittent period (p < 0.05). The wiVD and wiPD were lower in the PSS-affected eyes than in the fellow eyes and in the control eyes in the PSS-attacked period (p < 0.05). All segmental VD and PD was lower in the PSS affected eyes than in the healthy control eyes (p < 0.05). In intermittent period, the wiVD and wiPD were lower in the PSS-affected eyes than in the fellow eyes (p < 0.05). Age, CCT, and SSI were associated with macular wiVD and wiPD in PSS attacked period. Age and CCT were associated with macular wiVD and wiPD in PSS intermittent period. CONCLUSION: Decreased macular superficial VD and PD was found in patients with Posner-Schlossman syndrome in attacked period and in remission. Macular wiVD and wiPD were associated with age, CCT and SSI in PSS patients.

Indication of the prognosis of pulmonary hypertension by using CMR function parameters.

OBJECTIVE: This study aimed to compare the cardiac function among different sub-types of pulmonary hypertension (PH) and to explore the independent predictors of major adverse cardiovascular events (MACE). METHODS: Eighty-seven PH patients diagnosed by right heart catheterization (RHC) were recruited. Patients underwent cardiac magnetic resonance (CMR) and RHC examination within 2 weeks. The CMR images were analyzed to calculate the cardiac functional parameters including right ventricle (RV) and left ventricle (LV) end-diastolic volume index (EDVI), end-systolic volume index (ESVI), stroke volume index (SVI), ejection fraction (EF), tricuspid annular plane systolic excursion (TAPSE), and myocardial mass (MM). The median follow-up time was 46.5 months (interquartile range: 26-65.5 months), and the endpoints were the occurrence of MACE. RESULTS: RVEDVI, LVEDVI, and LVESVI were higher in congenital heart disease-related PH (CHD-PH) than in other sub-types (p < 0.05). RVMM, RVSVI, and RVCI were highest in CHD-PH. There was no significant difference in the prognosis among different sub-types (p > 0.05). Comparing with the non-MACE group, RVEF, TAPSE, and LVSVI significantly decreased in the MACE group, while the RVESVI significantly increased (p < 0.05). TAPSE ≤ 15.65 mm and LVSVI ≤ 30.27 mL/m2 were significant independent predictors of prognosis in PH patients. CONCLUSION: CHD-PH had a higher RV function reserve but lowest LVEF comparing to other subgroups. TAPSE and LVSVI could contribute to the prediction of MACE in PH patients. KEY POINTS: • CMR imaging is a noninvasive and accurate tool to assess ventricular function. • CHD-PH had higher RV function reserve but lowest LVEF. • TAPSE and LVSVI could contribute to the prediction of MACE in PH patients.

Impact of obstructive sleep apnea complicated with type 2 diabetes on long-term cardiovascular risks and all-cause mortality in elderly patients.

BACKGROUND: The prognostic significance of obstructive sleep apnea (OSA) in elderly patients with type 2 diabetes is unclear. The aim of this study was to determine the risk of cardiovascular disease (CVD) and mortality in elderly patients with OSA complicated with type 2 diabetes compared to patients with OSA without type 2 diabetes. METHODS: From January 2015 to October 2017, 1113 eligible elderly patients with OSA, no history of cardiovascular, ≥60 years of age, and complete follow-up records were enrolled in this consecutive multicentre prospective cohort study. All patients had completed polysomnography (PSG) examinations. An apnoea-hypopnoea index of ≥5 events per hour recorded by polysomnography was defined as the diagnostic criterion for OSA. We collected baseline demographics, clinical characteristics, sleep parameters and follow-up outcomes. The primary aim of this study was to identify the risk of incident major adverse cardiovascular events (MACE). Secondary outcomes were all-cause mortality, components of MACE and a composite of all events. Kaplan-Meier survival analysis and Cox proportional hazards models were used to evaluate whether type 2 diabetes was associated with incident events. RESULTS: A total of 266 (23.9%) patients had OSA complicated with type 2 diabetes. MACE occurred in 97 patients during the median 42-month follow-up. Kaplan-Meier survival curves indicated a significant relationship between type 2 diabetes and MACE (log-rank P = 0.003). Multivariable Cox regression analysis showed that type 2 diabetes increased the risk of MACE (HR = 1.64, 95% CI:1.08-2.47, P = 0.019), hospitalisation for unstable angina (HR = 2.11, 95% CI:1.23-3.64, P = 0.007) and a composite of all events in elderly patients with OSA (HR = 1.70, 95% CI:1.17-2.49, P = 0.007). However, there were no significant differences in the incidence of cardiovascular death, all-cause mortality, MI and hospitalisation for heart failure between patients with and without diabetes (P > 0.05). The subgroup analysis demonstrated that females (AHR = 2.46, 95% CI:1.17-5.19, P = 0.018), ≥ 70 years (AHR = 1.95, 95% CI:1.08-3.52, P = 0.027), overweight and obese (AHR = 2.04, 95% CI:1.29-3.33, P = 0.002) with mild OSA (AHR = 2.42, 95% CI: 1.03-5.71, P = 0.044) were at a higher risk for MACE by diabetes. CONCLUSION: OSA and type 2 diabetes are interrelated and synergistic with MACE, hospitalisation for unstable angina and a composite of all events development. Overweight and obese females, ≥ 70 years with mild OSA combined with type 2 diabetes presented a significantly high MACE risk.

miRNA and circRNA expression patterns in mouse brain during toxoplasmosis development.

BACKGROUND: Increasing evidence has shown that circular RNAs (circRNAs) are involved in neurodegenerative disorders, but their roles in neurological toxoplasmosis are yet to know. This study examined miRNA and circRNA expressions in mouse brain following oral infection with T. gondii Pru strain. RESULTS: Total RNA extracted from acutely infected (11 days post infection (DPI)), chronically infected (35 DPI) and uninfected mouse brain samples were subjected to genome-wide small RNA sequencing. In the acutely infected mice, 9 circRNAs and 20 miRNAs were upregulated, whereas 67 circRNAs and 28 miRNAs were downregulated. In the chronically infected mice, 2 circRNAs and 42 miRNAs were upregulated, whereas 1 circRNA and 29 miRNAs were downregulated. Gene ontology analysis predicted that the host genes that produced the dysregulated circRNAs in the acutely infected brain were primarily involved in response to stimulus and ion binding activities. Furthermore, predictive interaction networks of circRNA-miRNA and miRNA-mRNA were constructed based on genome-wide transcriptome sequencing and computational analyses, which might suggest the putative functions of miRNAs and circRNAs as a large class of post-transcriptional regulators. CONCLUSIONS: These findings will shed light on circRNA-miRNA interactions during the pathogenesis of toxoplasmosis, and they will lay solid foundation for studying the potential regulation roles of miRNAs and circRNAs in T. gondii induced pathogenesis.

InAs/GaAs quantum dot narrow ridge lasers epitaxially grown on SOI substrates for silicon photonic integration.

Monolithic integration of III-V laser sources on standard silicon-on-insulator (SOI) substrate has been recognized as an enabling technology for realizing Si-based photonic integration circuits (PICs). The Si-based ridge lasers employing III-V quantum dot (QD) materials are gaining significant momentum as it allows massive-scalable, streamlined fabrication of Si photonic integrated chips to be made cost effectively. Here, we present the successful fabrication of InAs/GaAs QD ridge lasers monolithically grown on {111}-faceted SOI hollow substrates. The as-cleaved Fabry-Perot (FP) narrow ridge laser is achieved with a relatively low threshold current of 50 mA at room temperature under pulse current operation. The maximum working temperature achieved is up to 80 oC. The promising lasing characteristics of such SOI-based InAs/GaAs QD ridge lasers with low threshold current and small footprint provide a viable route towards large-scale, low-cost integration of laser sources on SOI platform for silicon photonic integration purpose.

Phosphorus-free 1.5 µm InAs quantum-dot microdisk lasers on metamorphic InGaAs/SOI platform.

III-V semiconductor lasers epitaxially grown on silicon, especially on a silicon-on-insulator (SOI) platform, have been considered one of the most promising approaches to realize an integrated light source for silicon photonics. Although notable achievements have been reported on InP-based 1.5 µm III-V semiconductor lasers directly grown on silicon substrates, phosphorus-free 1.5 µm InAs quantum dot (QD) lasers on both silicon and SOI platforms are still uncharted territory. In this work, we demonstrate, to the best of our knowledge, the first phosphorus-free InAs QD microdisk laser epitaxially grown on SOI substrate emitting at the telecommunications S-band by growing metamorphic InAs/InGaAs QDs on (111)-faceted SOI hollow structures. The lasing threshold power for a seven-layer InAs QD microdisk laser with a diameter of 4 µm is measured as 234 µW at 200 K. For comparison, identical microdisk lasers grown on GaAs substrate are also characterized. The results obtained pave the way for an on-chip 1.5 µm light source for long-haul telecommunications.

Bioconversion of indole-3-acetonitrile by the N2-fixing bacterium Ensifer meliloti CGMCC 7333 and its Escherichia coli-expressed nitrile hydratase.

An N2-fixing bacterium, Ensifer meliloti CGMCC 7333, has been reported to degrade the cyano-containing neonicotinoid insecticides acetamiprid and thiacloprid using a nitrile hydratase (NHase). Here, the bioconversion of indole-3-acetonitrile (IAN) by E. meliloti, Escherichia coli overexpressing the NHase, and purified recombinant NHase was studied. E. meliloti converted IAN to the product indole-3-acetamide (IAM), and no nitrilase or amidase activities, or indole-3-acetic acid formation, were detected. Whole cells of E. meliloti converted IAN from the initial content of 6.41 to 0.06 mmol/L in 48 h. Meanwhile, forming 5.99 mmol/L IAM, the molar conversion of 94.4%. E. coli Rosetta overexpressing the NHase from E. meliloti produced 4.46 mmol/L IAM in 5 min, with a conversion rate of 91.1%. The purified NHase had a Vmax for IAN conversion of 294.28 U/mg. Adding 2% and 10% (v/v) dichloromethane to 50 mmol/L sodium phosphate buffer containing 200 mg/L IAN increased the NHase activity by 26.8% and 11.5% respectively, while the addition of 20% hexane had no inhibitory effect on IAN bioconversion. E. meliloti shows high NHase activity without forming a byproduct carboxylic acid, and its tolerance of dichloromethane and hexane increases its potential for application in the green biosynthesis of high-value amide compounds.

Aphananoid A is an Anti-Inflammatory Limonoid with a New 5/6/5 Fused Ring Featuring a C24 Carbon Skeleton from Aphanamixis polystachya.

Aphananoid A, a limonoid which features a rare C24 appendage and new 5/6/5 fused-ring framework, was obtained from Aphanamixis polystachya. The planar structure as well as the absolute configuration was identified based on extensive spectroscopic analysis and electronic circular dichroism calculations. The biogenetic pathway of aphananoid A was also speculated, which arises from the triterpene by the 3,4-seco-7,8-seco-6,8 cyclo-7,30-decarbon key pattern. In addition, bioassays indicated that aphananoid A inhibited NO production in the RAW264.7 cell line (46.80 ± 1.93%).

[Association of copy number of SMN1 and SMN2 with clinical phenotypes in children with spinal muscular atrophy].

OBJECTIVE: To study the association of copy number of SMN1 and SMN2 with clinical phenotypes in children with spinal muscular atrophy (SMA). METHODS: A total of 45 children with SMA were enrolled. Multiplex ligation-dependent probe amplification was used to measure the gene copy numbers of SMN1 and SMN2. The association of copy number of SMN1 and SMN2 with clinical phenotypes was analyzed. RESULTS: Of the 45 children with SMA, 42 (93%) had a homozygous deletion of SMN1 exons 7 and 8, and 3 (7%) had a deletion of SMN1 exon 7 alone. No association was found between SMA clinical types and the deletion types of SMN1 exons 7 and 8 (P>0.05). There was a significant difference in the distribution of SMN2 gene copy numbers between the children with SMA and the healthy children (P<0.05). The children with SMA usually had two or three copies of SMN2 gene, while the healthy children usually had one or two copies of SMN2 gene. There was a significant difference in the distribution of SMN2 copy numbers among the children with different SMA clinical types (P<0.05). The children with two copies of SMN2 gene had a significantly lower age of onset than those with three or four copies. Most of the children with type I SMA had two or three copies of SMN2 gene. Most of the children with type II SMA had three copies of SMN2 gene. Most of the children with type III SMA had three or four copies of SMN2 gene. Children with a higher copy number of SMN2 gene tended to have an older age of onset and better motor function and clinical outcome, and there was a significant association between SMN2 gene copy number and clinical outcome (P<0.05). CONCLUSIONS: The SMN2 gene can reduce the severity of SMA via the dosage compensation effect. SMN2 copy number is associated with the phenotype of SMA, and therefore, it can be used to predict disease severity.

δ-Selective Functionalization of Alkanols Enabled by Visible-Light-Induced Ligand-to-Metal Charge Transfer.

We demonstrate the application of ligand-to-metal charge transfer (LMCT) excitation to the direct catalytic generation of energetically challenging alkoxy radicals from alcohols through a coordination-LMCT-homolysis process with an abundant and inexpensive cerium salt as the catalyst. This catalytic manifold provides a simple and efficient way to utilize the characteristic reactivity and selectivity of transient alkoxy radicals for δ-selective C-H bond functionalization. Under mild redox-neutral conditions without the need for prefunctionalization, this method provides a versatile platform to access molecular complexity from simple and abundant alcohols.

Cerium-Catalyzed Formal Cycloaddition of Cycloalkanols with Alkenes through Dual Photoexcitation.

We describe a synergistic utilization of cerium photocatalysis and photoinduced electron transfer catalysis that enables an atom- and step-economical ring expansion of readily available cycloalkanols. This operationally simple protocol provides rapid access to privileged and synthetically challenging bridged lactones. The mild catalytic manifold has been adapted to continuous flow for scale-up applications and employed for the concise synthesis of polycyclic core of nepalactones.

The Reference Intervals for Serum C-Terminal Agrin Fragment in Healthy Individuals and as a Biomarker for Renal Function in Kidney Transplant Recipients.

BACKGROUND: C-terminal agrin fragment (CAF) has been shown to be a promising new biomarker for kidney function. The aim of this study was to verify the reference intervals for CAF in Chinese healthy adults and to assess the efficiency of CAF for monitoring renal function after transplantation. METHODS: Serum samples were collected from 200 healthy adult subjects and 60 living donor kidney recipients before and on day 1, day 2 and at 6 months after transplantation. We measured serum CAF, creatinine, cystatin C and NGAL concentrations at each time. Estimated glomerular filtration rate (eGFR) was evaluated by Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. Reference intervals for CAF were determined at 2.5th and 97.5th percentiles. RESULTS: Serum CAF concentrations were observed to be higher in females of old age groups while no significant differences were discovered in males between age groups. There were significant gender-related differences in CAF in old age groups (50-64 and ≥65 years). Serum CAF correlated positively with serum creatinine, cystatin C and negatively with eGFR on day 1, day 2 and at 6 months after kidney transplantation. CAF and NGAL fell rapidly into the normal range on the second postoperative day, prior to creatinine and cystatin C. CONCLUSIONS: This study verified the reference intervals for serum CAF. CAF could be a potential new biomarker for kidney function monitoring.

Serum Neutrophil Gelatinase-Associated Lipocalin and Cystatin C for Assessing Recovery of Graft Function in Patients Undergoing Living-Donor Kidney Transplantation.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) can be used as an early indicator of acute kidney injury (AKI), and cystatin C is also suggested to be an ideal marker of glomerular filtration rate (GFR), but they were not sufficiently studied in recipients without delayed graft function (DGF) after living-donor kidney transplant (LDKT). The aim of the study is to investigate whether serum NGAL and cystatin C can assess the recovery of renal function after LDKT. METHODS: 49 adult patients that had undergone LDKT between January 2012 and March 2014 were prospectively enrolled. Serum creatinine, NGAL, and cystatin C were measured on day 0-7, day 10, day 14 and month 9 after transplant. Recovery of graft function was evaluated by the time needed to reach eGFR > or = 60 mL/min/1.73 in2 Poor long-term graft outcome was defined as eGFR < 60 mL/min/1.73 M2 at 9 months. RESULTS: No DGF was recorded. Serum NGAL level decreased to normal earlier than creatinine after transplant. Cystatin C declined rapidly, but still stayed above the normal range. Serum NGAL on day 0 (p = 0.028) and cystatin C on day 2 (p < 0.001) were independent predictors of the time for graft function recovery in multivariate analysis. Compared to recipients with fair long-term graft outcome (eGFR > 60 mL/min/1.73 m2 at 9 months), recipients with poor long-term graft outcome (eGFR < 60 mL/min/1.73 m2 at 9 months) displayed higher serum NGAL on day 2 (p = 0.045), older age (p = 0.002), longer time on dialysis (p = 0.02), and lower donor eGFR (p = 0.045). There were correlations between serum NGAL and eGFR on day 0 and day 2. Correlations between serum cystatin C and eGFR on day 0, day 2, and month 9 were all significant. CONCLUSIONS: Serum NGAL may be used as an early predictor of recovery of post-transplant graft function after LDKT, but may not be used for real-time assessment of GFR. At the same time, the predictive ability of serum cystatin C needs to be further assessed.