Genetic diversity and population structure of Echinococcus multilocularis: An in-silico global analysis.

Objective: Alveolar echinococcosis is caused by Echinococcus multilocularis, a parasite of zoonotic significance with a wide range of intermediate and final hosts, and the parasite survives successfully in diversified conditions. Plentiful studies have been done to study the genetic structure of the population of the parasite and the level of intimate kinship using mitochondrial (mt) DNA. The present study was conducted to investigate the population structure, genetic variation, and phylogenetic relationship of various isolates of E. multiocularis submitted to GenBank worldwide. Sequences of mt genes (mt-cytochrome c oxidase (cox1), mt-NADH dehydrogenase (nad1)) of E. multilocularis were analyzed to achieve the set goals. Materials and Methods: A total of 275 and 124 gene sequences of mt-cox1 and mt-nad1 belonging to E. multilocularis, respectively, were retrieved from the National Center for Biotechnology Information GenBank. The retrieved sequences were subjected to alignment with respective reference sequences using MEGA software. The PopArt software was used to establish median-joining networks, while DnaSp was used to calculate neutrality and diversity indices. MrBayes software was used to investigate the phylogenetic association between haplotypes based on Bayesian phylogeny. Results: Approximately 13 and 20 distinctive haplotypes of nad1 and cox1 genes, respectively, were observed in the present study. In both of the mt genes, diversity indices indicated low haplotype (mt-cox1 = 0.140; mt-nad1 = 0.374) and nucleotide (mt-cox1 = 0.00111; mt-nad1 = 0.00287) diversities. The values of Tajima's D and Fu Fs for a population of both of the genes under study were found to be negative. Conclusion: This study is a maiden attempt to provide insights into the population structure and genetic variation of E. multilocularis on a global scale. However, it is suggested that to better understand the population structure and genetic diversity of E. multilocularis, more geographical locations and amplifications of full-length gene sequences should be considered, which could be helpful in widening the insights into the genetic diversity of E. multilocularis.

Cultivating a greener future: Exploiting trichoderma derived secondary metabolites for fusarium wilt management in peas.

This study aimed to identify efficient Trichoderma isolate(s) for the management of Fusarium wilt in peas. Four different pea germplasms (Sarsabz, Pea-09, Meteor and Supreme) were evaluated for resistance against Fusarium oxysporum in pot assay. Resistant germplasm exhibits a varying range of disease severity (23%) and percent disease index (21%), whereas susceptible and highly susceptible germplasm exhibit maximum disease severity (44-79%) and percent disease index (47-82%). The susceptible germplasm Meteor was selected for in vivo experiment. Five different Trichoderma spp. (Trichoderma koningii, T. hamatum, T. longibrachiatum, T. viride, and T. harzianum) were screened for the production of hydrolytic extracellular enzymes under in vitro. In-vitro biocontrol potential of Trichoderma spp. was assayed by percentage inhibition of dry mass of Fusarium oxysporum pisi (FOP) with Trichoderma spp. metabolite filtrate concentrations. Maximum growth inhibition was observed by T. harzianum (50-89%). T. harzianum metabolites in filtrate conc. (40%, 50%, and 60%) exhibited maximum reduction in biomass and were thus used for in vivo management of the disease. The pot experiment for in-vivo management also confirmed the maximum inhibition of FOP by T. harzianum metabolites filtrate at 60% by reducing disease parameters and enhancing growth, yield, and physiochemical and stress markers. Trichoderma strains led to an increase in chlorophyll and carotenoids (34-26%), Total phenolic 55%, Total protein content 60%, Total Flavonoid content 36%, and the increasing order of enzyme activities were as follows: CAT > POX > PPO > PAL in all treatments. These strains demonstrate excellent bio-control of Fusarium wilt in pea via induction of defense-related enzymes. The present work will help use Trichoderma species in disease management programme as an effective biocontrol agent against plant pathogens.

Cleft Palate in a Newborn With Trisomy 21: A Case Report.

Trisomy 21, or Down syndrome (DS), is neonates' most common chromosomal abnormality. In addition, children born with DS have an increased risk of congenital anomalies such as congenital heart defects, gastrointestinal abnormalities, and, rarely, cleft palate. Cleft lip and palate are among the most common congenital anomalies associated with many congenital syndromes; however, Trisomy 21 is the least common congenital anomaly associated with orofacial clefts. We present a case of cleft palate, duodenal stenosis, persistent pulmonary hypertension of the newborn, patent ductus arteriosus, and atrial septal defect in a newborn with classical clinical features of Down syndrome. This report discusses the uncommon presentation of trisomy 21 and concomitant cleft palate in a neonate, including its recognition and treatment, as no standard of care treatment exists.

Nickel toxicity pretreatment attenuates salt stress by activating antioxidative system and ion homeostasis in tomato (Solanum lycopersicon L.): an interplay from mild to severe stress.

Plants antioxidative system is the first line of defense against oxidative stress caused secondarily by toxic ions under salinity. Plants with pre-activated antioxidative system can better adapt to salinity and can result in higher growth and yield. The current experiment was conducted to assess the adaptation of two tomato genotypes (Riogrande and Green Gold) with pre-activated antioxidative enzymes against salt stress. Tomato seedlings were exposed to mild stress (Ni: 0, 15 and 30 mg L-1) for three weeks to activate the antioxidative enzymes. The seedlings with pre-activated antioxidative enzymes were then grown under severe stress in hydroponics (0, 75 and 150 mM NaCl) and soil (control, 7.5 and 15 dS m-1) to check the adaptation, growth and yield. The results showed that Ni toxicity significantly enhanced activities of antioxidant enzymes (SOD, CAT, APX and POX) in both the genotypes and reduced growth with higher values in genotype Riogrande than Green Gold. The seedlings with pre-activated antioxidant enzymes showed better growth, low Na+ and high K+ uptake and maintained higher antioxidative enzymes activity than non-treated seedlings after four weeks of salt stress treatment in hydroponics. Similarly, the results in soil salinity treatment of the Ni pretreated seedlings showed higher yield characteristics (fruit yield per plant, average fruit weight and fruit diameter) than non-treated seedlings. However, Ni pretreatment had nonsignificant effect on tomato fruit quality characteristics like fruit dry matter percentage, total soluble solids, fruit juice pH and titratable acidity. The genotype Riogrande showed better growth, yield and fruit quality than Green Gold due to higher activity of antioxidant enzymes and better ion homeostasis as a result of Ni pretreatment. The results suggest that pre-activation antioxidant enzymes by Ni treatment proved to be an effective strategy to attenuate salt stress for better growth and yield of tomato plants.

Synthesis and antibacterial potential of Loranthus pulverulentus conjugated silver nanoparticles.

The green method is not only harmonious to our environment but saves time and is comparatively cheaper than other methods. The current study is aimed to synthesize the silver nanoparticles (AgNPs) using the green method by using the leaves of Loranthus pulverulentus Wall (LPW) which functioned as a reducing as well as capping agent. The synthesis of biogenic AgNPs was confirmed by UV-Vis spectroscopy (UV-Vis.), Scanning electron microscopy (SEM), X-ray diffraction (XRD), energy dispersive X-ray (EDX), and Fourier transform infrared (FTIR). The absorbance maxima of the synthesized AgNPs were observed in the range of 405-435 nm. The SEM was used to find out the size and shape of the synthesized AgNPs, which revealed that the NPs were rectangular having sizes from 05 to 16 nm. XRD revealed that the synthesized NPs were crystalline and face-centered cubic (FCC). EDX analysis reflected the elemental composition of the synthesized product and showed that the percentage of silver = 92. FTIR was used to confirm the functional groups responsible for the reduction and stabilization of silver ions. It revealed that biomolecules present in plant extract were responsible for the reduction and stabilization of silver ions. The effect of temperature, pH, reaction time, silver nitrate concentration, and plant extract concentration on the synthesis of AgNPs were also investigated. The synthesized silver nanoparticles were tested against four bacterial strains which showed strong antibacterial activity. We conclude that biogenic silver nanoparticles could be used as antibacterial agents to treat various diseases. RESEARCH HIGHLIGHTS: Loranthus pulverulentus Wall leaf extract mediated synthesis of silver nanoparticles. Effects of pH 3, 4, 6, 9, and 11, reaction time 5, 10, 20, 40 min, 1, 2, and 3 h, temperature 30, 40, 50, 60°C, silver nitrate concentration 0.5, 1.0, 2.0, and 3.0 mM and plant extract concentration 0.5, 1.0, 2.0, and 3.0 g on the AgNPs synthesis were also studied Scanning electron microscopy analysis revealed a small size of synthesized silver nanoparticles (8 nm). The synthesized silver nanoparticles were found effective against various bacterial pathogens.

An Emerged Challenge of Air Pollution and Ever-Increasing Particulate Matter in Pakistan; A Critical Review.

This study, for the first time, aims to evaluate the situation of air quality in Pakistan critically; through a detailed assessment of sources, policies, and key challenges to identify the plausible way forward. Air pollution and particulate matter have merged as a global challenge in recent years because of its growing health and socio-economic risks. The intensity and impacts of these risks have become more pronounced, especially in developing countries like Pakistan that lack adequate warning, protection, and management systems. Various epidemiological studies have linked poor air quality with different health disorders and increasing death rates. In Pakistan, mortality rates as a result of exposure to increased levels of air pollutants, especially particulate matter, are alarming. According to the World Bank's estimates, Pakistan's annual burden of disease from outdoor air pollution is responsible for around 22,000 premature adult deaths and 163,432 DALYs (disability-adjusted life years) lost. The concentration of major air pollutants in Pakistan, such as NOx, O3, and SO2 have also been increasing significantly over the last two decades. Several studies are also reporting multiple instances of air quality around the major cities of Pakistan being consistently exceeding the national guidelines. During teh year 2019 PM2.5 cocnentrations in the city of Lahore revealed that almost every single day was in exceedance of the WHO and national air quality standards. Although the substantial effects of these rising pollutant concentrations in Pakistan have been stated in a few studies, however, the total extent, nature of contributing factors, and consequences remain inadequately understood. This study aims to use data available from monitoring stations, satellites, and literature to highlight the gaps in our understanding and emphasize the critical challenges associated with poor air quality faced in Pakistan. Topmost is the lack of air quality monitoring systems followed by poor initiatives by policymakers and enforcement agencies. A way forward while addressing these key challenges is also discussed, which focuses on the development of flexible monitoring, new technologies, and monitoring approaches in addition to communications among the various public, private agencies, and all relevant stakeholders.

In-silico pharmacophoric and molecular docking-based drug discovery against the Main Protease (Mpro) of SARS-CoV-2, a causative agent COVID-19.

COVID-19 (Coronavirus Disease 2019) caused by a novel 'SARS-CoV-2' virus resulted in public health emergencies across the world. An effective vaccine to cure this virus is not yet available, thus requires concerted efforts at various scales. In this study, we employed Computer-Aided Drug Design (CADD) based approach to identify the drug-like compounds - inhibiting the replication of the main protease (Mpro) of SARS-CoV-2. Our database search using an online tool "ZINC pharmer" retrieved ~1500 compounds based on pharmacophore features. Lipinski's rule was applied to further evaluate the drug-like compounds, followed by molecular docking-based screening, and the selection of screening ligand complex with Mpro based on S-score (higher than reference inhibitor) and root-mean-square deviation (RMSD) value (less than reference inhibitor) using AutoDock 4.2. Resultantly, ~200 compounds were identified having strong interaction with Mpro of SARS-CoV-2. After evaluating their binding energy using the AutoDock 4.2 software, three compounds (ZINC20291569, ZINC90403206, ZINC95480156) were identified that showed highest binding energy with Mpro of SARS-CoV-2 and strong inhibition effect than the N3 (reference inhibitor). A good binding energy, drug likeness and effective pharmacokinetic parameters suggest that these candidates have greater potential to stop the replication of SARS-CoV-2, hence might lead to the cure of COVID-19.

Investigating the impact of Glyoxal retrieval from MAX-DOAS observations during haze and non-haze conditions in Beijing.

This study presents the Multi Axis Differential Optical Absorption Spectroscopy (MAX-DOAS) measurements for Glyoxal (CHOCHO) in Beijing, China (39.95°N, 116.32°E). CHOCHO is the smallest compound of di-carbonyl group. As a primary sink of CHOCHO, its photolysis with NOx (oxides of nitrogen) results in the production of tropospheric ozone. Therefore, the focus of CHOCHO DOAS measurements is increasing in trend. We did the measurements from 09 May 2017 to 09 September 2017. The study was conducted to compare different retrieval settings in order to reveal best DOAS fit settings for CHOCHO; furthermore, effect of haze and non-haze days on CHOCHO concentration was examined. The root mean square of residual and Differential Slant Column density (dSCD) error was reduced when measurements were done with lower wavelength limit around 432-438 nm and upper intervals around 455-460 nm. Thus, lower wavelength intervals around 432-438 nm and upper intervals around 457-460 nm were best for the retrieval of dSCDs for CHOCHO. Meteorological conditions like haze or non-haze days did not have significant effect on DOAS fit parameters. The CHOCHO vertical column densities range from 1.33E+14 to 9.77E+14 molecules/cm2 during the study period with average of 6.16E+14 molecules/cm2. The results indicated that during haze days CHOCHO concentration was higher because of lower rate of photolysis and atmospheric oxidation potential. Our results did not show any significant weekend effect on CHOCHO atmospheric concentration.

Evidence of genetically diverse virulent mating types of Phytophthora capsici from Capsicum annum L.

Chili pepper (Capsicum annum L.) is an important economic crop that is severely destroyed by the filamentous oomycete Phytophthora capsici. Little is known about this pathogen in key chili pepper farms in Punjab province, Pakistan. We investigated the genetic diversity of P. capsici strains using standard taxonomic and molecular tools, and characterized their colony growth patterns as well as their disease severity on chili pepper plants under the greenhouse conditions. Phylogenetic analysis based on ribosomal DNA (rDNA), ß-tubulin and translation elongation factor 1α loci revealed divergent evolution in the population structure of P. capsici isolates. The mean oospore diameter of mating type A1 isolates was greater than that of mating type A2 isolates. We provide first evidence of an uneven distribution of highly virulent mating type A1 and A2 of P. capsici that are insensitive to mefenoxam, pyrimorph, dimethomorph, and azoxystrobin fungicides, and represent a risk factor that could ease outpacing the current P. capsici management strategies.

Invasive Aspergillus terreus morphological transitions and immunoadaptations mediating antifungal resistance.

BACKGROUND AND AIMS: Aspergillus terreus Thom is a pathogen of public health and agricultural importance for its seamless abilities to expand its ecological niche. The aim of this study was holistically to investigate A. terreus morphological and immunoadaptations and their implication in antifungal resistance and proliferation during infection. MATERIALS AND METHODS: In-depth unstructured mining of relevant peer-reviewed literature was performed for A. terreus morphological, immune, resistance, and genetic diversity based on the sequenced calmodulin-like gene. RESULTS: Accessory conidia and phialidic conidia produced by A. terreus confer discrete anti-fungal resistance that ensures survivability during therapies. Interestingly, by producing unique metabolites such as Asp-melanin and terretonin, A. terreus is capable of hijacking macrophages and scavenging iron, respectively. As such, A. terreus has established a rare mechanism to mitigate phagocytosis and swing the interaction dynamics in favor of its proliferation and survival in hosts. CONCLUSION: It is further unraveled that besides A. terreus genetic diversity, morphological, biochemical, and immunologic adaptations associated with conidia germination and discharge of chemical signals during infection enable masking of the host defense as an integral part of its strategy to survive and rapidly colonize hosts.

Biofortification of Wheat Cultivars to Combat Zinc Deficiency.

Zinc (Zn) deficiency caused by inadequate dietary intake is a global nutritional problem, particularly in developing countries. Therefore, zinc biofortification of wheat and other cereal crops is being urgently addressed and highly prioritized as a research topic. A field study was planned to evaluate the influence of zinc application on grain yield, grain zinc content, and grain phytic acid concentrations of wheat cultivars, and the relationships between these parameters. Three wheat cultivars, C1 = Faisalabad-2008, C2 = Punjab-2011, and C3 = Millet-2011 were tested with five different methods of zinc application: T1 = control, T2 = seed priming, T3 = soil application, T4 = foliar application, and T5 = soil + foliar application. It was found that grain yield and grain zinc were positively correlated, whereas, grain phytic acid and grain zinc were significantly negatively correlated. Results also revealed that T5, T3, and T4 considerably increased grain yield; however, T2 only slightly enhanced grain yield. Grain zinc concentration increased from 33.1 and 33.7 mg kg-1 in T1 to 62.3 and 63.1 mg kg-1 in T5 in 2013-2014 and 2014-2015, respectively. In particular, T5 markedly decreased grain phytic acid content; however, maximum concentration was recorded in T1. Moreover, all the tested cultivars exhibited considerable variation in grain yield, grain zinc, and grain phytic acid content. In conclusion, T5 was found to be most suitable for both optimum grain yield and grain biofortification of wheat.

Interaction of formaldehyde with a water-tolerant frustrated Lewis pair.

A facile complexation of formaldehyde with the water-tolerant frustrated Lewis pair (FLP) B(C6F5)3/PtBu3 and its Al-analog under ambient conditions is reported. Unprecedented formaldehyde adducts 1, 2 and 4 have been identified and crystallographically characterized.

Transparent Films from CO2 -Based Polyunsaturated Poly(ether carbonate)s: A Novel Synthesis Strategy and Fast Curing.

Transparent films were prepared by cross-linking polyunsaturated poly(ether carbonate)s obtained by the multicomponent polymerization of CO2 , propylene oxide, maleic anhydride, and allyl glycidyl ether. Poly(ether carbonate)s with ABXBA multiblock structures were obtained by sequential addition of mixtures of propylene oxide/maleic anhydride and propylene oxide/allyl glycidyl ether during the polymerization. The simultaneous addition of both monomer mixtures provided poly(ether carbonate)s with AXA triblock structures. Both types of polyunsaturated poly(ether carbonate)s are characterized by diverse functional groups, that is, terminal hydroxy groups, maleate moieties along the polymer backbone, and pendant allyl groups that allow for versatile polymer chemistry. The combination of double bonds substituted with electron-acceptor and electron-donor groups enables particularly facile UV- or redox-initiated free-radical curing. The resulting materials are transparent and highly interesting for coating applications.

Anion effect controlling the selectivity in the zinc-catalysed copolymerisation of CO2 and cyclohexene oxide.

The choice of the anion has a surprisingly strong effect on the incorporation of CO2 into the polymer obtained during the zinc-catalysed copolymerisation of CO2 and cyclohexene oxide. The product span ranges from polyethercarbonates, where short polyether sequences alternate with carbonate linkages, to polycarbonates with a strictly alternating sequence of the repeating units. Herein, we report on the influence of the coordination ability of the anion on the selectivity and kinetics of the copolymerisation reaction.

Are parents really attached to their adopted children?

BACKGROUND: Psychological studies found that adopted children suffer from lack of attachment relationships in life. It is important for new parents to understand the underlying concepts before they begin to comprehend behavior issues arising out of different turbulent situations in an adopted child's life. Attachment theory facilitates in comprehending the frame of mind of these children, when they come from emotionally turbulent backgrounds and how some, if not all behavior issues can be attempted to be resolved to recognize children better and to create a nurturing relationship between adopted child and new parents. FINDINGS: Focus group method was deployed to collect the data via un-restricted non-probability sampling approach; data was quantified for evaluating the hypotheses via t-test of equality of means. Cross cultural findings suggested that parents-adopted children relationship in terms of secure attachment is revealed more in non-working parents, female parents, children of 11-14 years and female children across stated nations while, the ambivalent, avoidant and disorganized attachments are found more in practice if parents are working & male parents and if foster children are male at large & of 15-18 years. CONCLUSION: It is concluded that the task of creating an enriched attachment relationship with an adopted child depends more on parents, normally non working parents and female parents while quality time and care is given somehow the other to young and female kids by either of the parents for establishing quality attachment. Quality time being bestowed to kids translates the category and intensity of parents- children associations.

Asian ascendancy: media in the age of globalization.

This paper aims to investigate the media industry in Asia, with reference to international standards of media tools, instruments, content, and coverage. We have also explored factors that may further improve Asian media. We have used an empirical approach. Our findings revealed that the media in Asian significantly contribute to expanding cultural knowledge and the exchange of multilateral dialogues. However, they do not look after the interests of minorities or non-dominating communities. Although the media should be a virtual ambassador, they often provoke hostilities within regions. Governments own most media outlets in the developing nations in Asia, and so the media rely on government backing and are subject to restrictions. International and national regulations connected to media freedom or constraints should be explored to protect Asian societies. The practical implications of these negative aspects are that the Asian media does not help the plights of minorities or minimize the fear of war in the region. The universal lesson of brotherhood among humanity for all colors and races should be preached by the media. In this paper, we have concentrated on how Asian media influence cultural expansion, the exchange of multilateral dialogues, the interests of minorities, aggression between nations, and generate income for common citizens.

Digital scrapbooking as a standard of care in neonatal intensive care units: initial experience.

In this article, we describe a digital photo scrapbooking project as a standard of care for the parents of infants admitted in a neonatal intensive care unit (NICU). Photographs were taken from birth until discharge or expiry at special moments during the infant's hospitalization and used to create a digital scrapbook with daily notes by the parents. The scrapbook and original photos were provided on a CD at discharge or at expiry. Parents and their families unanimously appreciated the photos and the opportunity to record their thoughts, and considered the CDs as a lifetime treasure. Digital photo journaling could be implemented as a standard of care at other institutions with a commitment from the nursing and ancillary staff of the NICU and labor and delivery department, with possible support from volunteers.

A new one-pot hydroformylation/Strecker synthesis as a versatile synthetic tool for polyfunctional compounds and functionalization of dendrimers.

A versatile and high yielding one-pot hydroformylation/Strecker synthesis is reported for the synthesis of various alpha-aminonitriles. This methodology allows functionalization of dendrimers (e.g. polyamines 4-7) and hyper branched polymers (e.g. polyallyl glycerol 12) to give corresponding dendritic structures with alpha-aminonitriles and/or amino acids in the outer shell in good to excellent yields.

Al-Awadi/Raas-Rothschild/Schinzel (AARRS) phocomelia syndrome: case report and developmental field analysis.

We describe a girl infant with anomalies of the left pelvis and lower limb (pelvic, femoral, and tibial hypogenesis with absent fibula), subtle facial changes, patent foraman ovale, single umbilical artery, single kidney, and imperforate anus. The external genitalia were asymmetric and ambiguous with normal uterus and ovaries visualized by ultrasound. The anomalies are compatible with previously reported cases of Al-Awadi/Raas-Rothschild/Schinzel (AARRS) phocomelia, an autosomal recessive disorder with WNT7 gene mutations documented in one family. We suggest that AARRS phocomelia, Fuhrmann syndrome, and similar conditions comprise a spectrum, and that the anomaly pattern derives from serial action of the same signal pathways within primary (e.g., the major axes), secondary (e.g., heart or limb primordia), and/or local (e.g., tibial-fibular differentiation) developmental fields.

Sorptive potential of sunflower stem for Cr(III) ions from aqueous solutions and its kinetic and thermodynamic profile.

The sorptive potential of sunflower stem (180-300mum) for Cr(III) ions has been investigated in detail. The maximum sorption (>/=85%) of Cr(III) ions (70.2muM) has been accomplished using 30mg of high density sunflower stem in 10min from 0.001M nitric and 0.0001M hydrochloric acid solutions. The accumulation of Cr(III) ions on the sorbent follows Dubinin-Radushkevich (D-R), Freundlich and Langmuir isotherms. The isotherm yields D-R saturation capacity X(m)=1.60+/-0.23mmolg(-1), beta=-0.00654+/-0.00017kJ(2)mol(-2), mean free energy E=8.74+/-0.12kJmol(-1), Freundlich sorption capacity K(F)=0.24+/-0.11molg(-1), 1/n=0.90+/-0.04 and of Langmuir constant K(L)=6800+/-600dm(3)mol(-1) and C(m)=120+/-18micromolg(-1). The variation of sorption with temperature (283-323K) gives DeltaH=-23.3+/-0.8kJmol(-1), DeltaS=-64.0+/-2.7Jmol(-1)K(-1) and DeltaG(298k)=-4.04+/-0.09kJmol(-1). The negative enthalpy and free energy envisage exothermic and spontaneous nature of sorption, respectively. Bisulphate, Fe(III), molybdate, citrate, Fe(II), Y(III) suppress the sorption significantly. The selectivity studies indicate that Cr(III), Eu(III) and Tb(III) ions can be separated from Tc(VII) and I(I). Sunflower stem can be used for the preconcentration and removal of Cr(III) ions from aqueous medium. This cheaper and novel sorbent has potential applications in analytical and environmental chemistry, in water decontamination, industrial waste treatment and in pollution abatement. A possible mechanism of biosorption of Cr(III) ions onto the sunflower stem has been proposed.