Experimental and Theoretical Insights into Interfacial Properties of 2D Materials for Selective Water Transport Membranes: A Critical Review.

Interfacial properties, such as wettability and friction, play critical roles in nanofluidics and desalination. Understanding the interfacial properties of two-dimensional (2D) materials is crucial in these applications due to the close interaction between liquids and the solid surface. The most important interfacial properties of a solid surface include the water contact angle, which quantifies the extent of interactions between the surface and water, and the water slip length, which determines how much faster water can flow on the surface beyond the predictions of continuum fluid mechanics. This Review seeks to elucidate the mechanism that governs the interfacial properties of diverse 2D materials, including transition metal dichalcogenides (e.g., MoS2), graphene, and hexagonal boron nitride (hBN). Our work consolidates existing experimental and computational insights into 2D material synthesis and modeling and explores their interfacial properties for desalination. We investigated the capabilities of density functional theory and molecular dynamics simulations in analyzing the interfacial properties of 2D materials. Specifically, we highlight how MD simulations have revolutionized our understanding of these properties, paving the way for their effective application in desalination. This Review of the synthesis and interfacial properties of 2D materials unlocks opportunities for further advancement and optimization in desalination.

Densitometric method for assessment of six specialized metabolites in four Sida sp. and its congener Abutilon indicum: Targeted metabolomics, greenness assessment, and chemometrics analysis.

Sida is one of the most diverse genera, with about 200 species distributed in tropical and subtropical regions of the world. Among 18 species distributed in India, Sida acuta, Sida cordifolia, Sida rhombifolia, and Sida cordata are used in traditional medicines along with its possible adulterant Abutilon indicum for several therapeutic uses. The non-availability of marker-based validated methods for the identification and classification of these species leads to adulteration. Indoloquinoline and quinazoline are the major bioactive alkaloids distributed in Sida spp. First time, a simple, economical and high throughput method was developed and validated for the simultaneous determination of 20-hydroxyecdysone (1), vasicine (2), vasicinone (3), cryptolepine (4), quindolinone (5), and cryptolepinone (6) using HPTLC-UV densitometry. The method was validated to meet globally accepted ICH guidelines. The method was sensitive with LOD and LOQ ranging from 0.38-0.63 and 1.57-2.12 µg/band. The samples were spiked at 3 different concentrations, the recovery values were 93.49-98.88%. In addition, the greenness index of the HPTLC method was estimated using four different greenness assessment techniques. Targeted HPTLC analysis indicated the distribution of specialized metabolites in Sida spp. and A. indicum. However, the occurrence of cryptolepine in A. indicum was not reported in the literature, so this was further confirmed by liquid chromatographic studies of the samples from different locations. The chromatographic data was statistically evaluated by principal component analysis (PCA) and hierarchical clustering (HCA). HPTLC-based targeted metabolite quantitation explains the adulteration/substitution in Sida raw material and derived herbal preparations.

Protrusio acetabuli in tubercular hip: Outcome of femoral head as autologous bone graft used during total hip replacement.

Purpose: Here we present a study of a tuberculosis hip protrusion with a large acetabular defect treated by total hip replacement using a novel technique without the use of any protrusio ring, wire mesh, or cage. To our knowledge, such a technique has not been described in the literature previously. Methodology: 25 patients with a past treatment history of hip tuberculosis came to our medical college with complaints of worsening pain in the hip joint and limping. The hip pain and limping were progressive in nature and became more severe, and his affected lower limb shortened. Results: The average AK distance (horizontal distance between Kohlar line and the medial acetabular border) before surgery was 8 mm (range: 1-16). For the acetabular implant, the mean abduction angle was 45° (range: 41-48), and the mean anteversion angle was 18° (range: 13-24). The average preoperative shortening for limb length inequality was 4 mm (range: 0-11). The average HHS (Harris Hip Score) during follow-up was 87.6 points. Conclusion: In TB hip protrusio, an autograft of the femur head is used to fill the acetabular defect and provide a good approach for these patients without any extra economic burden or complications as seen in a metallic ring or cage.

Reduction of esters to alcohols and iodides using aminodiborane (µ-NH2B2H5): scope and mechanistic investigations.

Herein, we report an efficient methodology for the reduction of esters, carbonates, and anhydrides to alcohols using in situ generated aminodiborane from iodine and ammonia borane. This methodology also finds use for the transformation of esters to iodides by varying the stoichiometry of reagents. The protocol has broad substrate scope for transformation of esters to alcohols and iodides with excellent yields. The method is also useful for synthesizing pharmaceutically and industrially important compounds such as a Cinacalcet precursor, a Streptoindole analogue, and 1,4-pentanediol. Control studies and DFT calculations carried out to study the reduction mechanism of esters using aminodiborane indicate that a dioxaborinamine intermediate is formed during the reaction.

C-H Bond Functionalization of Aryl Acids and Amines by 'On-water' Reaction: Bi-dentate Directing Group Enabled Synthesis of Biaryl and m-Teraryl Carboxamides.

Herein, we report a palladium-catalyzed 'on-water' methodology for the synthesis of biaryl and m-teraryl derivatives of aryl carboxamides by selective mono and bis C-H bond functionalization. 8-aminoquinoline and 2-thiomethylaniline were used as directing groups for C-H bond functionalization of aryl carboxamides with various aryl and alkyl iodides using 3.0-4.0 mol% of Pd(OAc)2 as catalyst and water as the solvent resulting in 45-97% isolated yields of the mono and bis C-H bond functionalized products. Using an 8-aminoquinoline carboxamide core, C-H bond functionalization of indole-3-carboxylic acid and a late-stage functionalization of aspirin molecule have also been carried out. Reactions of benzyl/naphthyl picolinamides with aryl iodides gave 60-96% yield of the arylated products using the picolinamide directing group. Moreover, the insoluble nature of products in an aqueous medium enabled us to explore the reusability of the solvent, i. e., water and the catalyst. Control experiments and structural studies were carried out which confirmed the in situ formation of unique palladacycles during the reaction. Removal of the directing groups has been carried out to convert the functionalized products into amines and fluorenone compounds useful in industrial and pharmaceutical applications.

A Bench-stable 8-Aminoquinoline Derived Phosphine-free Manganese (I)-Catalyst for Environmentally Benign C(α)-Alkylation of Oxindoles with Secondary and Primary Alcohols.

Herein, we report a new air-stable phosphine-free 8-AQ (8-aminoquinoline) based Mn(I) carbonyl complex as the catalyst for the C(α)-alkylation of oxindoles with alcohols. The Mn complex [(8-AQ)Mn(CO)3 Br] works effectively as a catalyst for the α-alkylation of oxindoles by both secondary as well as primary alcohols. The procedure has been used for the synthesis of pharmaceutically important recently developed oxindoles such as 3-(4-methoxybenzyl)indolin-2-one, 3-(4-(dimethylamino)benzyl)indolin-2-one, 3-(4-(dimethylamino)phenyl)-5-fluoroindolin-2-one and 3-(benzo[d][1,3]dioxol-5-ylmethyl)indolin-2-one, which are found to be effective in preventing specific types of cell death in neurodegenerative disorders. Control experiments have been carried out to investigate the reaction mechanism and the crucial role of metal-ligand cooperation via -NH2 moiety during catalysis.

Stimulation of glucose uptake by glycosides from Alectra parasitica subsp. chitrakutensis (M.A. Rau) K.K. Khanna & An. Kumar: An in-vitro study of TNF-α induced insulin resistance in L6 myoblasts.

Alectra parasitica subsp. chitrakutensis (M.A. Rau) K.K. Khanna & An. Kumar (Orobanchaceae) is a parasitic plant indigenous to India. Locally, the plant is known as 'Midaki and Nirgundikand'. It is used to treat fever, piles, cardiovascular disorders, and blood-borne non-infectious diseases by ethnic communities. The phytochemical investigation of A. parasitica subsp. chitrakutensis rhizome led to the isolation of azafrin (1), rehmaionoside-C (2), and mussaenoside (3). Compounds (2) and (3) are being reported for the first time from this plant. Compounds were evaluated for their intercellular glucose uptake activity in basal and insulin-TNF-α-stimulated L6 muscle cells. In particular, rehmaionoside C exhibited activity comparative to metformin, increasing uptake by basal- and insulin-TNF-α-stimulated cells by 4.88- and 3.90-fold and 5.04- and 4.04-fold. While azafrin and mussaenoside have produced 3.03- and 2.36-fold; 4.03- and 3.22-fold increase in intercellular glucose uptake. Compounds did not show toxicities in rat L6 myoblast cells. The study suggests that rehmaionoside-C from A. parasitica subsp. chitrakutensis might activate glucose uptake by insulin mimics and could be a nontoxic anti-diabetes lead for drug discovery.

Surface Roughness Explains the Observed Water Contact Angle and Slip Length on 2D Hexagonal Boron Nitride.

Hexagonal boron nitride (hBN) is a two-dimensional (2D) material that is currently being explored in a number of applications, such as atomically thin coatings, water desalination, and biological sensors. In many of these applications, the hBN surface comes into intimate contact with water. In this work, we investigate the wetting and frictional behavior of realistic 2D hBN surfaces with atomic-scale defects and roughness. We combine density functional theory calculations of the charge distribution inside hBN with free energy calculations using molecular dynamics simulations of the hBN-water interface. We find that the presence of surface roughness, but not that of vacancy defects, leads to remarkable agreement with the experimentally observed water contact angle of 66° on freshly synthesized, uncontaminated hBN. Not only that, the inclusion of surface roughness predicts with exceptional accuracy the experimental water slip length of ∼1 nm on hBN. Our results underscore the importance of considering realistic models of 2D materials with surface roughness while modeling nanomaterial-water interfaces in molecular simulations.

Advanced Meditation and Vegan Diet Increased Acylglycines and Reduced Lipids Associated with Improved Health: A Prospective Longitudinal Study.

Introduction: Samyama is an Isha Yoga 8-day residential meditation/yoga retreat combined with 60 days of preparation with vegan diet. We showed earlier Samyama retreat was associated with lower systemic inflammation and favorable lipid profiles along with other physical and mental health benefits. There is no mechanistic study on the impact of an advanced meditative process on multiple blood lipids and their implications on meditation-related improved physical and mental wellbeing. Methods: Sixty-four Samyama participants on vegan diet had blood sampled immediately before and immediately after the 8-day retreat for lipidomic analysis. The complex plasma lipidome was characterized using high-resolution mass spectrometric analysis and tandem mass spectrometry. Results: Pre- and post-Samyama blood samples of 64 Samyama participants were analyzed. Acylglycines (acetyl, propionyl, butyryl, and valeryl) were increased in the plasma post-Samyama compared with pre-Samyama (p < 0.001). Levels of glycerophosphocholines, glycerophosphoethanolamines, di-unsaturated ethanolamine plasmalogens, cholesterol esters, acylcarnitines, and acylgylcerines (triacylglycerols and diacylglycerols) decreased after the Samyama meditation. Plasma levels of glycerophosphoserines or glycerophosphoinositols were unchanged. Conclusion: An 8-day advanced meditation retreat resulted in increased acylglycines, an endocannabinoid-like fatty acid amide associated with increased cellular anandamide levels, anti-inflammation, analgesia, and vascular relaxation. Other serum lipid levels, including some that are associated with increased risk of atherosclerosis, were reduced following the Samyama program. ClinicalTrials.gov Registration: Identifier: NCT04366544.

Detection of pathogenic Leptospira spp. in herpetofauna in Central Appalachia.

Leptospirosis is a water borne zoonotic disease of global significance that is caused by pathogenic species of the genus Leptospira. Pathogenic leptospires live in the kidneys of reservoir or infected animals and are shed in their urine contaminating water, soil, etc. Rodents are considered the primary reservoir of leptospirosis, but little is known about the role of herpetofauna (non-avian reptiles and amphibians) in the epidemiology of the disease. To address this, various species of amphibians and reptiles in the Cumberland Gap Region of the Central Appalachia were screened for the presence of Leptospira spp. Kidneys harvested from of a total of 116 amphibians and reptiles belonging to seven species of snakes, seven species of salamanders, seven species of frogs/toads, seven species of turtles and one species of lizards were tested using a highly specific TaqMan based qPCR that targets lipl32 gene of pathogenic Leptospira spp. Overall, 15 of the tested 116 amphibians and reptiles were positive (12.9%; 95% CI: 7.4%-20.4%). Of the 101 amphibians, 11 were positive (10.9%; 95% CI: 5.6%-18.7%), and 4 of the 15 reptiles tested positive (26.7%; 95% CI: 7.8%-55.1%). The amplified gene fragments of lipl32 from qPCR positive kidneys were sequenced and found to be identical with known pathogenic Leptospira spp. These results suggest that although the proportion of reptiles and amphibians transmitting pathogenic Leptospira spp. within the environment may be low as compared to rodents, they pose a risk to other susceptible hosts that share their habitats and may have role in maintaining a baseline infection in the environment.

Electrochemical biosensor for serogroup specific diagnosis of leptospirosis.

A problem with the current leptospirosis diagnostic methods is the low sensitivity and specificity during the acute phase of illness. Rapid point-of-care (POC) assays with minimal sample utilization and low cost are desired in clinical practice. Here, we report for the first time lipopolysaccharide (LPS) based electrochemical biosensor that offers a rapid, highly sensitive, serogroup specific diagnosis of leptospirosis during the acute stage of infection and also to distinguish from other flu like infections. The proposed sensor is fabricated by the immobilization of LPS onto dodecanethiol (DT) modified gold electrode. Monolayer of DT is attached through covalent bond (Au-S) interaction onto the gold electrode. Thus, leptospiral antibodies from the human serum samples bind to the LPS present on self-assembled monolayer (SAM) of DT and showed a higher RCT value compared to SAM. The detection limit of the developed LPS sensor is estimated to be 100 nM. This biosensor is the first electrochemical sensing platform used for detection of LPS from Leptospira spp. This method is completely a solution-based diagnostic method and therefore it is rapid, simple, and sensitive; thus establishing a key technology towards a useful POC diagnostic strategy in serogroup level and hence an alternative to MAT.

Occurrence of potentially zoonotic and cephalosporin resistant enteric bacteria among shelter dogs in the Central and South-Central Appalachia.

BACKGROUND: Antimicrobial resistance and presence of zoonotic enteropathogens in shelter dogs pose a public health risk to shelter workers and potential adopters alike. In this study we investigated the prevalence of zoonotic bacterial pathogens and cephalosporin resistant (CefR) enteric bacteria in the feces of apparently healthy shelter dogs in the Cumberland Gap Region (CGR) in the US states of Kentucky, Tennessee and Virginia. RESULTS: Fecal samples of 59 dogs from 10 shelters in the CGR of Central and South-Central Appalachia were screened for the presence of Campylobacter jejuni, Clostridium perfringens, Salmonella and CefR enteric bacteria. C. jejuni, C. perfringens were detected by PCR based assays. Culture and PCR were used for Salmonella detection. Of 59 dogs, fecal samples from 14 (23.7%) and 8 (13.6%) dogs tested positive for cpa and hipO genes of C. perfringens and C. jejuni, respectively. Salmonella was not detected in any of the tested samples by PCR or culture. CefR enteric bacteria were isolated on MacConkey agar supplemented with ceftiofur followed by identification using MALDI-TOF. Fecal samples from 16 dogs (27.1%) yielded a total of 18 CefR enteric bacteria. Majority of CefR isolates (14/18, 77.8%) were E. coli followed by, one isolate each of Enterococcus hirae, Acinetobacter baumannii, Acinetobacter pittii, and Pseudomonas aeruginosa. CefR enteric bacteria were tested for resistance against 19- or 24-antibiotic panels using broth microdilution method. Seventeen (94.4%) CefR bacteria were resistant to more than one antimicrobial agent, and 14 (77.8%) displayed multidrug resistance (MDR). CONCLUSIONS: This study shows that shelter dogs within the CGR not only carry zoonotic bacterial pathogens, but also shed multidrug resistant enteric bacteria in their feces that may pose public health risks.

Associations among Stress, Anxiety, Depression, and Emotional Intelligence among Veterinary Medicine Students.

Background: Veterinary students are faced with immense pressures and rigors during school. These pressures have contributed to elevated levels of stress, anxiety, and depression (SAD) among veterinary students relative to the general population. One proposed concept to help students combat SAD is that of emotional intelligence (EI). We explored the relationship between EI and SAD among veterinary students at a college in the Southeast United States. Methods: A cross-sectional study design was implemented among a convenience sample of 182 veterinary medical students. The survey instrument contained 56 items that elicited information about students' demographics, perceived stress, anxiety, and depression, and emotional intelligence levels. Data analysis included univariate statistics, Pearson's correlations, and multiple regression and independent samples t-tests. Results: The study revealed a statistically significant, negative correlation between EI levels and stress, anxiety, and depression. Additionally, a statistically significant, positive correlation was found between stress and anxiety as well as both stress and anxiety and depression. Multiple linear regression showed that EI was a statistically significant predictor of stress (b = -0.239, p < 0.001), anxiety (b = -0.044, p < 0.001), and depression (b = -0.063, p < 0.001), after controlling for sociodemographic variables. Students' t-test results revealed a statistically significant mean difference in EI scores among students screening positive versus negative for depression, with students screening negative having a mean EI score of 10.81 points higher than students who screened positive for depression. Conclusion: There is a scientifically supported need for interventions in veterinary school to integrate EI into the veterinary medical curriculum and consider the EI levels of veterinary student candidates.

Biochemical analysis of leptospiral LPS explained the difference between pathogenic and non-pathogenic serogroups.

Lipopolysaccharide (LPS) is the major surface antigen of Leptospira. In this study, the genes involved in the LPS biosynthesis were analyzed and compared by bioinformatics tools. Also, the chemical composition analysis of leptospiral lipopolysaccharides (LPS) extracted from 5 pathogenic serovars like Autumnalis, Australis, Ballum, Grippotyphosa, Pomona, and the nonpathogenic serovar Andamana was performed. Methods used were Limulus amebocyte lysate assay (LAL), gas chromatography-mass spectrometry (GC-MS), fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR). LAL assay showed a significantly higher level of endotoxicity among pathogenic serovars (~0.490 EU/mL) than that of nonpathogenic Andamana (~0.102 EU/mL). FAMES analysis showed the presence of palmitic acid (C16:0), hydroxy lauric acid (3-OH-C12:0), and oleic acid (C18:0). Palmitoleic acid (C16: 1), and 3- hydroxy palmitate (3-OH-C16:0) was detected only in pathogenic serovars. In contrast myristoleic acid (C14:1) and stearic acid (C18:0) were present in Andamana. FTIR analysis revealed C-O-C stretch of esters, 3°ROH functional groups and carbohydrate vibration range were similar among pathogenic serovars. The NMR analysis reveals similarity for 6 deoxy sugars and methyl groups of Autumnalis, Australis, and Ballum. Further, the presence of palmitoleic acid and 3-hydroxy palmitate may be the significant pathogen-associated predisposing factor. This mediates high osmolarity glycerol (HOG) mediated stress response in leptospiral LPS mediated pathogenesis.

Translation and validation of the Test of Adherence to Inhalers (TAI) questionnaire among adult patients with asthma in Malaysia.

OBJECTIVE: The Test of Adherence to Inhalers (TAI) is a validated self-reported questionnaire that can reliably assess adherence to inhalers through patient self-report. However, it was not available in Bahasa Melayu (BM) language, nor has it been validated for use in the Malaysian population. The study aimed, therefore, to translate the 10-item TAI questionnaire into BM and evaluate its psychometric properties. METHODS: The translation of the English version of the valid 10-item TAI questionnaire into BM was followed by subjecting it to a series of tests establishing factorial, concurrent and known group validities. Concurrent validity was assessed through Spearman's rank correlation coefficient against pharmacy refill-based adherence scores. Known group validity was assessed by cross-tabulation against asthma symptom control and using chi-square test. The internal consistency of the test scale was determined by a test-retest method using Cronbach's alpha (α) value and intraclass correlation coefficients. RESULTS: A total of 120 adult asthma patients participated in the study. A 2-factor structure was obtained and confirmed with acceptable fit indices; CFI, NFI, IFI, TLI >0.9 and, RMSEA was 0.08. The reliability of the scale was 0.871. The test-retest reliability coefficient for the total sum score was 0.832 (p < 0.01), which indicated good reliability. The 10-item TAI-BM established concurrent and known group validities. The sensitivity and specificity of the tool were >85%. CONCLUSIONS: The scale successfully translated into BM and validated. The 10-item TAI-BM appears fit for use in testing inhaler adherence of Malaysian patients with asthma.

Arsenic-induced differential expression of oxidative stress and secondary metabolite content in two genotypes of Andrographis paniculata.

The present study explores the differential responses of two genotypes (APwC: wild collection and APMS: mass selection line) of A. paniculata against the three application rates of arsenic (42, 126, and 200 mg kg-1). The oxidative enzymes, As accumulation in different tissues, plant growth, and content of pharmacologically important ent-labdane-related diterpenes (ent-LRDs) of the two genotypes were evaluated in the study. Results demonstrated that As uptake significantly reduced plant biomass in APwC and APMS by 5-41.5% and 9-33% in a dose-response manner, respectively. The APMS exhibited lower bioconcentration and translocation factors, higher As tolerance index, and higher content of ent-LRDs as compared to APWC. As treatment induced a decrease in the sum of four metabolite content of APMS (1.43 times) and an increase in that of APWC (1.12 times) as compared to control. Likewise, variance in the production of 5,7,2',3'-tetramethoxyflavanone, and stress enzymes was also observed between APwC and APMS. The increase in the expression of ApCPS2 suggested its involvement in channeling of metabolic flux towards the biosynthesis of ent-LRDs under As stress.

Evaluation of Antimicrobial Stewardship Programs (ASPs) and their perceived level of success at Makkah region hospitals, Kingdom of Saudi Arabia.

Antimicrobial stewardship programs (ASPs) are collaborative efforts to optimize antimicrobial use in healthcare institutions through evidence-based quality improvement strategies. The general administration of pharmaceutical care in the Saudi ministry of health (MOH) is putting outstanding efforts in implementing antimicrobial stewardship in Saudi health care settings. Several surveys have been conducted globally and reported many types of antimicrobial stewardship strategies in health institutions and their effectiveness. This study aims to identify ASPs in Makkah region hospitals and their perceived level of success. We administered a regional survey to explore current progress and issues related to the implementation of ASPs in Makkah region hospitals at the pharmacy level (n = 25). Among responding hospitals, 19 (76%) hospitals, the most commonly reported ASP were as following: formulary restrictions (90%) for broad-spectrum antimicrobials and use of prospective feedback on antimicrobial prescribing (68%), use of clinical guidelines and pathways (100%), and use of automatic stop orders (68%) to limit inappropriate antimicrobial therapy. The study outcomes will also be of pivotal importance to devise policies and strategies for antimicrobial stewardship implementation in other non-MOH settings in the Makkah region. Based on our results, all reported institutions have at least one antimicrobial stewardship program in a process with a high success rate. A multidisciplinary ASP approach, active involvement of drug & therapeutic committee, formulary restrictions, and availability of education & training of pharmacists and physicians on ASP are the primary elements for perceived successful antimicrobial stewardship programs in the Makkah region hospitals.

Effectiveness of color and picture labeling in improving the knowledge on topical medications among patients with psoriasis: A randomized controlled trial.

AIM AND OBJECTIVE: The success of topical treatment in patients with psoriasis is still below par in Malaysia. The contributing factors include patients' understanding and knowledge about prescribed topical medications. The aim of this study was to assess the effectiveness of color and picture labeling (C and P labeling) in improving the knowledge about topical medications among patients with psoriasis. MATERIALS AND METHODS: An unblinded randomized controlled trial was undertaken at the dermatology clinic of a tertiary care hospital. Consent from the patients fulfilling inclusion criteria were obtained, and they were included in this study. They were randomized into two groups, namely Gp-1 (C and P labeling) and Gp-2 (conventional labeling). Both groups were assessed at week 0, 6, and 12 (visit 1, 2, and 3) using knowledge assessment list and psoriasis severity assessment score. For visit 2 (week 6), reinforcement of their understanding of topical treatment was performed. RESULTS: A total of 101 patients were recruited. Only 91 of them completed the study. The mean ages were 44.52 (±16.61) and 45.49 (±15.84) years, with 70.3% males and approximately half Malay ethnics. The changes of knowledge and comparison of Topical Application Assessment Score between the groups showed an incremental raise of significance with every visit (P = 0.006 [week 1], 0.004 [week 6], and 0.002 [week 12]). Psoriasis Area and Severity Index 75 could not draw any conclusion as patients who achieved >75% improvement were inadequate. CONCLUSION: C and P labeling was effective in improving the understanding and knowledge of patients with psoriasis. Both groups showed improvement in body surface area and Dermatology Life Quality Index for every visit; however, it was statistically insignificant.