Repetitive Transcranial Magnetic Stimulation (rTMS) Improves Cognitive Impairment and Intestinal Microecological Dysfunction Induced by High-Fat Diet in Rats.

Consuming a high-fat diet (HFD) is widely recognized to cause obesity and result in chronic brain inflammation that impairs cognitive function. Repetitive transcranial magnetic stimulation (rTMS) has shown effectiveness in both weight loss and cognitive improvement, although the exact mechanism is still unknown. Our study examined the effects of rTMS on the brain and intestinal microecological dysfunction. rTMS successfully reduced cognitive decline caused by an HFD in behavioral assessments involving the Y maze and novel object recognition. This was accompanied by an increase in the number of new neurons and the transcription level of genes related to synaptic plasticity (spindlin 1, synaptophysin, and postsynaptic protein-95) in the hippocampus. It was reached that rTMS decreased the release of high mobility group box 1, activation of microglia, and inflammation in the brains of HFD rats. rTMS also reduced hypothalamic hypocretin levels and improved peripheral blood lipid metabolism. In addition, rTMS recovered the HFD-induced gut microbiome imbalances, metabolic disorders, and, in particular, reduced levels of the microvirus. Our research emphasized that rTMS enhanced cognitive abilities, resulting in positive impacts on brain inflammation, neurodegeneration, and the microbiota in the gut, indicating the potential connection between the brain and gut, proposing that rTMS could be a new approach to addressing cognitive deficits linked to obesity.

Correction: Green synthesis of chlorella-derived carbon dots and their fluorescence imaging in zebrafish.

[This corrects the article DOI: 10.1039/D3RA07623G.].

A direct estrogenic involvement in the expression of human hypocretin.

Hypocretin is synthesized exclusively in the hypothalamus and distributes inputs to several areas of the brain, which may play an important role in depression. Our previous study showed that hypocretin-1 was increased in the lateral hypothalamus in female patients with depression compared to female controls. Estrogen acts through estrogen receptor (ER)α and ERß. We studied the possibility of a direct action of estrogen receptors on the expression of human hypocretin. We found that hypocretin-1 plasma levels were significantly higher in female patients with depression than in female controls. Female depression estrogen receptors and hypocretin are colocalized in the human lateral hypothalamus, PC12, and SK-N-SH cells. The estrogen receptor response elements (ERE) that exist in the hypocretin promoter region may directly regulate the gene expression of hypocretin. The synchronicity of change of hypocretin and estradiol both in hypothalamus and plasma was verified in female rats. In the presence of estradiol, specific binding occurs between the recombinant human ER and hypocretin-ERE. Expression of ER combined with estradiol repressed hypocretin promoter activity via the ERE. In conclusion, we found that estradiol may directly affect hypocretin neurons in the human hypothalamus via ER binding to the hypocretin-ERE, which may lead to the sex-specific pathogenesis of depression.

Smart Use of Skin Biopsy Punch in Treating Keloids: A Single-Center Retrospective Study.

BACKGROUND: Treatment of scarring has long been a problem due to high incidence and recurrence. Despite many existing treatment therapies, the efficacy remains unstable. OBJECTIVES: To determine the efficacy and safety of skin biopsy punch in combination with corticosteroid injection (BPCI) in treating keloids. APPROACH: This was a retrospective study. In total, 16 patients with keloids received BPCI. Changes in scar appearance, accompanied symptoms, and Vancouver Scar Scale (VSS) were analyzed. Patient satisfaction, VAS scores, and adverse effects were also evaluated. RESULTS: Scar appearance, accompanied symptoms, and VSS scores improved significantly after the treatment. The total effective rate was 93.75% at an 18-month follow-up on average. The mean reduction rate of VSS score was 58.44% (p < 0.0001), especially in height and pliability (84.44% and 78.19%, p < 0.0001). The recurrence rate in this study was 12.5% (n = 2) at an 18-month follow-up on average. Mild adverse effects of pain, pruritus, hypopigmentation, and telangiectasia were recorded. CONCLUSIONS: This study demonstrated BPCI might be an effective and safe therapy in keloids with a low long-time recurrence rate and well tolerance for patients. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these evidence-based medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Application of membrane separation technology in the purification of pharmaceutical components.

Traditional Chineae medicine (TCM) is often composed of a variety of natural medicines. Its composition is complex, and many of its components can not be analyzed and identified. The first step in the rational application of TCM is to successfully separate the effective components which is also a great inspiration for the development of new drugs. Among the many separation technologies of TCM, the traditional heating concentration separation technology has high energy consumption and low efficiency. As a new separation technology, membrane separation technology has the characteristics of simple operation, high efficiency, environment-friendly and so on. The separation effect of high molecular weight difference solution is better. The applications of several main membrane separation technologies such as microfiltration, nanofiltration, ultrafiltration and reverse osmosis are reviewed, the methods of restoring membrane flux after membrane fouling are discussed, and their large-scale industrial applications in the future are prospected and summarized.

Mating behaviors in ovoviviparous black rockfish (Sebastes schlegelii): molecular function of prostaglandin E2 as both a hormone and pheromone.

Prostaglandins (PGs) are profound hormones in teleost sexual behavior, especially in mating. PGs act as pheromones that affect the olfactory sensory neurons of males, inducing the initiation of a series of mating behaviors. However, the molecular mechanism by which PGs trigger mating behavior in ovoviviparous teleosts is still unclear. In the present study, we employed the ovoviviparous black rockfish (Sebastes schlegelii), an economically important marine species whose reproductive production is limited by incomplete fertilization, as a model species. The results showed that when the dose of PGE2 was higher than 10 nmol/L, a significant (P < 0.05) increase in mating behaviors was observed. Dual-fluorescence in situ hybridization indicated that PGE2 could fire specific neurons in different brain regions and receptor cells in the olfactory sac. After combining with specific neurons in the central nervous system (CNS), a series of genes related to reproduction are activated. The intracerebroventricular administration of PGE2 significantly increased lhb levels (P < 0.05) in both sexes. Moreover, steroidogenesis in gonads was also affected, inducing an increase (P < 0.05) in E2 levels in males and T levels in females. PGE2 levels were also increased significantly (P < 0.05) in both sexes. The present study revealed that PGE2 can activate mating behavior in black rockfish in both hormone and pheromone pathways, leading to variations in sex steroid levels and activation of reproductive behaviors. Our results provide not only novel insight into the onset of mating behaviors in ovoviviparous teleosts but also solutions for the incomplete fertilization caused by natural mating in cage aquaculture. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00214-w.

Fluorescent imaging and toxicology study of alga-derived carbon dots in zebrafish.

With the widespread application of carbon dots (CDs) in fluorescence imaging, their toxicity has become a focal point of concern. The potential toxicity of CDs synthesized from different raw materials remains an unresolved issue. Laver and wakame, which are commonly popular sea vegetable foods rich in nutrients, were utilized to investigate whether synthetic CDs derived from these alga sources retain medicinal value. Herein, two types of fluorescent alga-derived CDs were prepared through hydrothermal synthesis using laver and wakame respectively. Zebrafish were immersed in both types of CDs to observe their fluorescence imaging effects within the zebrafish bodies. It was observed that laver-derived CDs and wakame-derived CDs exhibited similar luminescence properties but differed in terms of fish egg imaging localization. Additionally, intestinal flora sequencing revealed varying degrees of influence on the zebrafish gut microbiota by the two types of CDs, suggesting that both alga-derived CDs could enhance the abundance of intestinal flora in zebrafish.

Green synthesis of chlorella-derived carbon dots and their fluorescence imaging in zebrafish.

Recently, carbon dots (CDs) have been shown to exhibit exceptional water solubility, low toxicity, favorable biocompatibility, stable fluorescence properties with a wide and continuous excitation spectrum, and an adjustable emission spectrum. Their remarkable characteristics make them highly promising for applications in the field of bioimaging. Zebrafish is currently extensively studied because of its high genetic homology with humans and the applicability of disease research findings from zebrafish to humans. Therefore, spirulina, a commonly used feed additive in aquaculture, was chosen as the raw material for synthesizing fluorescent CDs using a hydrothermal method. On the one hand, CDs can modulate dopamine receptors in the brain of zebrafish, leading to an increase in dopamine production and subsequently promoting their locomotor activity. On the other hand, CDs have been shown to enhance the intestinal anti-inflammatory capacity of zebrafish. This study aimed to explore the chronic toxicity and genotoxicity of CDs in zebrafish while providing valuable insights for their future application in biological and medical fields.

Bidirectional Catalysis Disintegration and Mineral Polymerization via Endogenous Iron(III) from Iron-Rich Sludge in Synergy with Waste Incineration Fly Ash.

To enhance the utilization of solid waste in cement kiln co-processing, this study analyzed the multifaceted synergy of pyrolysis and mineralization processes of iron-rich sludge (SS) and waste incineration fly ash (FA) at optimal blending ratios. Based on the physicochemical properties of SS and co-pyrolysis experiments, it was found that Fe acted as a positive catalyst in pyrolysis between 700 and 1000 °C, while the endogenous polymerization effect of Fe(III) mineral groups dominated above 800 °C. Additionally, the study investigated the solidification and migration of heavy metals and the transformation of harmful elements (S, Cl, and P). Results indicated that the best mixture ratios for SS and FA were 6:4 and 9:1, respectively, and synergistic pyrolysis and mineral co-curing effects were observed in the pyrolysis temperature range of 50-1000 °C. The synergy between SS and FA allowed for the decomposition and solidification of harmful organic components and heavy metals, reducing environmental risks. Furthermore, in actual production, by mixing 100 tons of SS and FA with Portland cement with a daily output of 2500 tons, the compressive strength during early hydration stages can reach 34.52 MPa on the third day, exceeding the highest performance of Portland cement (62.5R) strength index specified in the standard.

Evaluating the effect of food components on the digestion of dietary nucleic acids in human gastric juice in vitro.

Nucleic acids (NAs) were recently shown to be digested by pepsin in vitro; however, NAs digestion in human gastric juice in vivo is more complicated because of the complex gastric environment and ingestion of other food components. The purpose of this study was to investigate the digestibility of NAs in real human gastric juices after ingestion of other food components. As a result, DNA digestion was not affected when carbohydrates, proteins, and metal elements were ingested within the recommended dietary intake levels. Separately, protein exerted an inhibitory effect on DNA digestion when the mass ratio of protein:DNA was greater than 40:1. DNA exists in the nucleoprotein, which is closer to the state of DNA in real food, and was digested efficiently in human gastric juice. Meanwhile, DNA digestion was rarely affected even when the concentrations of monovalent ion (Na+) and divalent ions (Mg2+) were as high as 500 and 100 mM, respectively, and high concentration of Mg2+ ranged from 20 to 100 mM accelerated the digestion. In particular, short-stranded DNA (<100 nt) and miRNAs (19 ~ 25 nt) were not obviously degraded in human gastric juice. In conclusion, dietary NAs were digested efficiently and were not affected by other food components in human gastric juice, which may facilitate further digestion and utilization of DNA in the intestinal tract.

Functionalized nanopores based on hybridization chain reaction: Fabrication and microRNA sensing.

Enzyme-free hybridization chain reaction (HCR) technology is often used as a signal amplification tool for the detection of different targets. In this study, an ultrasensitive and label-free method for detecting miRNA-21 was developed using the nanopore ionic current rectification (ICR) technology coupled with HCR technology. The probe oligonucleotide (DNA1) was combined with the gold-coated nanopore through the Au-S bond to form a DNA1-functionalized gold-coated nanopore (DNA1-Au-coated nanopore). Since miRNA-21 is partially complementary to DNA1, it can be selectively recognized by DNA1-functionalized gold-coated nanopores. The target (miRNA-21) can induce the opening of hairpin DNA and HCR reaction after the introduction of hairpin DNA H1 and H2. The concentration of miRNA-21 will affect the combination of H1 and H2 on the inner wall of the nanopore, and its surface charge will change with the internal modification, thereby changing the ion current rectification ratio. Under the condition that the concentration of H1, H2 and HCR reaction time are constant, the change of ICR ratio is linearly correlated with the logarithm of miRNA-21 concentration within a certain range, which shows that the sensing strategy we designed can achieve target miRNA-21 detection. This ultrasensitive miRNA holds great promise in the field of cancer diagnosis.

Global trends and prospects in health economics of robotic surgery: a bibliometric analysis.

BACKGROUND: Over 10 million robotic surgeries have been performed. However, the cost and benefit of robotic surgery need to be evaluated to help hospitals, surgeons, patients, and payers make proper choices, making a health economic analysis necessary. The authors revealed the bibliometric profile in the field of health economics of robotic surgery to prompt research development and guide future studies. MATERIALS AND METHODS: The Web of Science Core Collection scientific database was searched for documents indexed from 2003 to 31 December 2022. Document types, years, authors, countries, institutions, journal sources, references, and keywords were analyzed and visualized using the Bibliometrix package, WPS Office software, Microsoft PowerPoint 2019, VOSviewer software (version 1.6.18), ggplot2, and Scimago Graphica. RESULTS: The development of the health economics of robotic surgery can be divided into three phases: slow-growing (2003-2009), developing (2010-2018), and fast-developing (2019-2022). J.C.H. and S.L.C. were the most active and influential authors, respectively. The USA produced the most documents, followed by China, and Italy. Korea had the highest number of citations per document. Surgical Endoscopy and Other Interventional Techniques accepted most documents, whereas Annals of Surgery, European Urology, and Journal of Minimally Invasive Gynecology had the highest number of citations per document. The Journal of Robotic Surgery is promising. The most-cited document in this field is New Technology and Health Care Costs - The Case of Robot-Assisted Surgery in 2010. The proportion of documents on urology is decreasing, while documents in the field of arthrology are emerging and flourishing. CONCLUSION: Research on the health economics of robotic surgery has been unbalanced. Areas awaiting exploration have been identified. Collaboration between scholars and coverage with provisions for evidence development by the government is needed to learn more comprehensively about the health economics of robotic surgery.

Effect of prebiotics, probiotics, synbiotics on depression: results from a meta-analysis.

Accumulating studies have shown the effects of gut microbiota management tools in improving depression. We conducted a meta-analysis to evaluate the effects of prebiotics, probiotics, and synbiotics on patients with depression. We searched six databases up to July 2022. In total, 13 randomized controlled trials (RCTs) with 786 participants were included. The overall results demonstrated that patients who received prebiotics, probiotics or synbiotics had significantly improved symptoms of depression compared with those in the placebo group. However, subgroup analysis only confirmed the significant antidepressant effects of agents that contained probiotics. In addition, patients with mild or moderate depression could both benefit from the treatment. Studies with a lower proportion of females reported stronger effects for alleviating depressive symptoms. In conclusion, agents that manipulate gut microbiota might improve mild-to-moderate depression. It is necessary to further investigate the benefits of prebiotic, probiotic and synbiotic treatments relative to antidepressants and follow up with individuals over a longer time before these therapies are implemented in clinical practice.

G-quadruplex-hemin DNAzyme functionalized nanopipettes: Fabrication and sensing application.

Solid-nanopores/nanopipettes have the exquisite ability to reveal the changes in molecular volume due to the advantages of adjustable size, good rigidity and low noise. Herein, a new platform for sensing application was established based on G-quadruplex-hemin DNAzyme (GQH) functionalized gold-coated nanopipettes. In this method, GQH was immobilized on gold-coated nanopipette, which could be used as a catalyst for the reaction of H2O2 with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) to promote the conversion of ABTS to ABTS+ ions inside gold-coated nanopipette, and the change of transmembrane ion current could be monitored in real time. At the optimal conditions, there was a correlation between the ion current and the concentration of H2O2 in a certain range, which could be used for the hydrogen peroxide sensing. The GQH immobilized nanopipette provides a useful platform to investigate enzymatic catalysis in confined environment, which can be used in electrocatalysis, sensing and fundamental electrochemistry.

Efficacy of Glucose Metabolism-Related Indexes on the Risk and Severity of Alzheimer's Disease: A Meta-Analysis.

BACKGROUND: Considering the strong correlation made between Alzheimer's disease (AD) and the pathology of glucose metabolism disorder, we sought to analyze the effects of fasting blood glucose (FBG) level, fasting plasma insulin (FINS) level, and insulin resistance index (HOMA-IR) on the risk and severity of AD. OBJECTIVE: Reveal the pathological relationship between AD and insulin resistance. METHODS: We searched 5 databases from inception through April 4, 2022. Meta-regression was conducted to identify if there were significant differences between groups. Shapiro-Wilk test and the Q-Q diagram were applied to evaluate the normality of variables. A multiple logistic regression model was employed to explore the association between FBG, FINS, HOMA-IR, and Mini-Mental State Examination scale score (MMSE). RESULTS: 47 qualified articles including 2,981 patients were enrolled in our study. FBG (p < 0.001), FINS (p < 0.001), and HOMA-IR (p < 0.001) were higher in AD patients than in controls. HOMA-IR was negatively correlated with MMSE (p = 0.001) and positively related to the sex ratio (male versus female) (p < 0.05). HOMA-IR obeyed lognormal distribution (p > 0.05), and the 95% bilateral boundary values were 0.73 and 10.67. FBG (p = 0.479) was positively correlated to MMSE, while FINS (p = 0.1657) was negatively correlated with MMSE. CONCLUSION: The increase in the levels of FBG, FINS, and HOMA-IR served as precise indicators of the risk of AD. HOMA-IR was found to be correlated to the increasing severity of AD, especially in male AD patients.

Prevalence and relevant factors of physical and emotional abuse by parents among children with autism spectrum disorder / 中国学校卫生

Objective@#To explore the prevalence and relevant factors of physical and emotional abuse by parents among children with autism spectrum disorder (ASD), so as to provide basis for intervention program of children abuse.@*Methods@#A total of 221 ASD children from 3 special education institutions in Tangshan were investigated from March to October in 2021, 395 non ASD children from two kindergartens in urban and rural areas were selected by convenient sampling. Parents of these children were invited for online and on site questionnaire survey. The self designed violence questionnaire, Childhood Autism Rating Scale and Patient Health Questionnaire-9 were used to assess violence, severity of autism, depression of parents. Chi square test, Fisher s exact probability method and Logistic regression were used to analyze the influencing factors of violence.@*Results@#About 81.9% of children with ASD and 72.9% of non ASD children experienced violence( P <0.05). The reported rates of physical and emotional violence in ASD children were 74.2% and 73.8% respectively, which in non ASD children were 58.7% and 65.8% respectively. There were significant differences in the 3 types of violence rate between the two groups ( P <0.05). Multivariate Logistic regression analysis showed that boys ( OR =1.70, 95% CI =1.12-2.60), annual per capita income <10 000 yuan( OR =2.43, 95% CI =1.45- 4.08 ), and parental depression ( OR mild =11.01, 95% CI =5.38-22.49; OR moderate =69.97，95% CI =24.25-201.93) were the risk factors for child violence exposure; ASD disease ( OR=1.96，95%CI =1.32-2.92), older age ( OR=1.19, 95%CI =1.01-1.41) and parental depression( OR mild =7.83, 95% CI =3.67-16.74; OR moderate =14.37，95% CI =6.17-33.46) were risk factors for physical violence; boys ( OR =1.62, 95% CI =1.11-2.36), mothers who work in manual labor ( OR=1.68, 95%CI =1.09-2.59) and parental depression ( OR mild =7.69, 95% CI =3.74-15.81; OR moderate =25.37, 95% CI =10.80-59.63) were risk factors for emotional violence( P < 0.05 ).@*Conclusion@#The reported rate of parental violence against children with ASD is high. Mental health promotion and social support for families with ASD should be strengthened.

Effect of Achievement Motivation and Self-Efficacy on General Well-Being among Students at Normal Universities in Ningxia: The Mediating Role of Time Management.

General well-being is a positive evaluation of one's mental health, which is an important topic in mental health. General well-being is fundamental to the positive development of young people. A thorough understanding of the factors that influence happiness have not yet been developed for students at normal universities in Ningxia Province in China. This study examined the mediation effects of time management on the relationship between achievement motivation, self-efficacy, and general well-being among students at normal universities in Ningxia Province in China. Using a random sampling, 163 participants (68 males and 95 females) completed the Achievement Motivation Scale (AMS), General Self-efficacy Scale (GSES), Time Management Questionnaire (TMQ) and General Well-being Scale (GWBS). Achievement motivation, self-efficacy, and time management were positively correlated with general well-being. The mediation effects of time management on the relationships between achievement motivation, self-efficacy, and general wellbeing (0.169 and 0.447) were demonstrated. These results add to the nuanced relationship between self-efficacy, achievement motivation, and general well-being. According to the Achievement Motivation Theory and the Self-efficacy Theory, this study reveals the role of achievement motivation, self-efficacy on general well-being and the mediating effect of time management in the relationship between achievement motivation, self-efficacy, and general well-being. According to the findings, the school can carry out a series of lectures and tutoring activities to enhance students' achievement motivation, self-efficacy, time management, and general well-being.

Stochastic Collision Electrochemistry from Single Pt Nanoparticles: Electrocatalytic Amplification and MicroRNA Sensing.

Single-particle collisions have made many achievements in basic research, but challenges still exist due to their low collision frequency and selectivity in complex samples. In this work, we developed an "on-off-on" strategy based on Pt nanoparticles (PtNPs) that catalyze N2H4 collision signals on the surface of carbon ultramicroelectrodes and established a new method for the detection of miRNA21 with high selectivity and sensitivity. PtNPs catalyze the reduction of N2H4 on the surface of carbon ultramicroelectrodes to generate a stepped collision signal, which is in the "on" state. The single-stranded DNA paired with miRNA21 is coupled with PtNPs to form the complex DNA/PtNPs. Because PtNPs are covered by DNA, the electrocatalytic collision of N2H4 oxidation is inhibited. At this time, the signal is in the "off" state. When miRNA21 is added, the strong complementary pairing between miRNA21 and DNA destroys the electrostatic adsorption of DNA/PtNP conjugates and restores the electrocatalytic performance of PtNPs, and the signal is in the "on" state again. Based on this, a new method for detecting miRNA21 was established. It provides a new way for small-molecule sensing and has a wide range of applications in electroanalysis, electrocatalysis, and biosensing.

Empathy experiences of Japanese certified genetic counselors: A qualitative investigation and proposed framework.

Empathy is an important element of genetic counseling. Most genetic counselors acknowledge the significance of empathically engaging clients. However, few empirical studies have focused on the empathy experience of genetic counselors, especially in non-Western countries. This study aimed to investigate Japanese genetic counselors' perspectives on the concept of empathy in clinical practice. The study conducted semi-structured interviews with Japanese certified genetic counselors who had approximately 10 years of clinical experience. Fourteen participants were interviewed about their thoughts on empathy and their experiences wherein they had deeply understood clients or felt closer to them. The interview data were analyzed using grounded theory. As a result, 17 categories were extracted, of which 13 were integrated into three themes of empathy: the empathic cycle in the relationships between genetic counselors and clients (cycling), the process of forming a deeper understanding of a client's perspectives (feeling), and the process of developing skills to understand clients with empathy (developing). The remaining four categories were grouped into the theme of "challenges of empathy." The categories included in the first three themes were similar to previous findings in Western countries, whereas some categories of challenges of empathy were unique to this study, which was conducted in a non-Western country. This might be attributed to the influence of Japanese culture, in which people emphasize self-regulation and an interdependent-self model. To our knowledge, this study is the first to report on Japanese certified genetic counselors' experiences of empathy. This study concludes with some suggestions for future research, including focusing on ways to overcome challenges of empathy in countries or healthcare systems.

Redescription and phylogenetic position of the terrestrial ciliates Gastrostylides dorsicirratus and Heterourosomoida lanceolata (Hypotricha, Dorsomarginalia).

The morphology and infraciliature of two soil hypotrichous ciliates, Heterourosomoida lanceolata (Shibuya, 1930) Singh and Kamra, 2015 and Gastrostylides dorsicirratus (Foissner, 1982) Foissner, 2016, were investigated using live observation and protargol staining. The Chinese population of H. lanceolata differs slightly from other populations in the body size in vivo, the relative length of the adoral zone, the number of right and left marginal cirri, the total number of dorsal bristles, and the number of micronuclei. The Chinese population of G. dorsicirratus corresponds well with Austrian and Indian populations. We also document the morphogenetic processes during binary fission of G. dorsicirratus, including the formation of the frontoventral-transverse cirral anlagen in a primary pattern. In addition, phylogenetic analyses based on SSU rDNA sequence data reveal that the two populations of G. dorsicirratus for which data are available cluster together with full support and form a clade with Heterourosomoida sinica and two populations of H. lanceolata.