A highly selective and sensitive rhodamine B-based chemosensor for Sn4+ in water-bearing and biomaging and biosensing in zebrafish.

A novel colorimetric-fluorescent dual-mode chemosensor (JT5) based on rhodamine B has been produced for monitoring Sn4+ in the DMSO/H2O (4:1, v/v) medium. It has high sensitivity, a low detection limit, a short response time (1 s) and high stability, and can still be maintained after two weeks with the red dual fluorescence/ colorimetric response. Enhancement of red fluorescence (591 nm) and red colorimetric (567 nm) response of JT5 by Sn4+ addition. The electrostatic potential of the sensor JT5 molecule was simulated to speculate on the sensing mechanism, and the IR, mass spectrometry and 1H NMR titration were utilized to further demonstrate that JT5 was coordinated to Sn4+ with a 1:1 type, the rhodamine spironolactam ring of JT5 opens up to form a penta-membered ring with Sn4+, meanwhile, its system may have chelation enhanced fluorescence (CHEF) effect. In addition, theoretical calculations were carried out to give the energy gaps of JT5 and [JT5 + Sn4+] as well as to simulate the electronic properties of the maximal absorption peaks. Notably, the sensor JT5 was successfully applied to monitoring Sn4+ in zebrafish, and the JT5-loaded filter paper provided a solid-state platform for detecting Sn4+ by both naked eye and fluorescent methods. In summary, this work contributes to monitoring Sn4+ in organisms and solid-state materials and promotes understanding of Sn4+ functions in biological systems, environments, and solid-state materials.

Tf2O-Mediated P(O)-N Bond Formation of Either P(O)-OH or P(O)-H Reagents with Multitype Amines.

We have developed a Tf2O-mediated approach for the direct amination of either P(O)-OH or P(O)-H reagents with a variety of aliphatic or aromatic amines. Without the requirement of precious metals and toxic reagents, this protocol provides an alternative route to various phosphinamides and phosphoramides. The reaction proceeds under simple and mild conditions and can be effectively scaled up with similar efficiency.

Air Quality and Health Implications of Coal Power Retirements Attributed to Industrial Electricity Savings in China.

The coal-dominated electricity system, alongside increasing industrial electricity demand, places China into a dilemma between industrialization and environmental impacts. A practical solution is to exploit air quality and health cobenefits of industrial energy efficiency measures, which has not yet been integrated into China's energy transition strategy. This research examines the pivotal role of industrial electricity savings in accelerating coal plant retirements and assesses the nexus of energy-pollution-health by modeling nationwide coal-fired plants at individual unit level. It shows that minimizing electricity needs by implementing more efficient technologies leads to the phaseout of 1279 hyper-polluting units (subcritical, <300 MW) by 2040, advancing the retirement of these units by an average of 7 years (3-16 years). The retirements at different locations yield varying levels of air quality improvements (9-17%), across six power grids. Reduced exposure to PM2.5 could avoid 123,100 pollution-related cumulative deaths over the next 20 years from 2020, of which ∼75% occur in the Central, East, and North grids, particularly coal-intensive and populous provinces (e.g., Shandong and Jiangsu). These findings provide key indicators to support geographically specific policymaking and lay out a rationale for decision-makers to incorporate multiple benefits into early coal phaseout strategies to avoid lock-in risk.

Flavomycin restores colistin susceptibility in multidrug-resistant Gram-negative bacteria.

Polymyxin is used as a last resort antibiotics for infections caused by multi-drug resistant (MDR) Gram-negative bacteria and is often combined with other antibiotics to improve clinical effectiveness. However, the synergism of colistin and other antibiotics remains obscure. Here, we revealed a notable synergy between colistin and flavomycin, which was traditionally used as an animal growth promoter and has limited activity against Gram-negative bacteria, using checkerboard assay and time-kill curve analyses. The importance of membrane penetration induced by colistin was assessed by examining the intracellular accumulation of flavomycin and its antimicrobial impact on Escherichia coli (E. coli) strains with truncated lipopolysaccharides. Besides, a mutation in the flavomycin binding site was created to confirm its role in the observed synergy. This synergy is manifested as an augmented penetration of the E. coli outer membrane by colistin, leading to increased intracellular accumulation of flavomycin and enhanced cell killing thereafter. The observed synergy was dependent on the antimicrobial activity of flavomycin, as mutation of its binding site abolished the synergy. In vivo studies confirmed the efficacy of colistin combined with flavomycin against MDR E. coli infections. This study is the first to demonstrate the synergistic effect between colistin and flavomycin, shedding light on their respective roles in this synergism. Therefore, we propose flavomycin as an adjuvant to enhance the potency of colistin against MDR Gram-negative bacteria. IMPORTANCE: Colistin is a critical antibiotic in combating multi-drug resistant Gram-negative bacteria, but the emergence of mobilized colistin resistance (mcr) undermines its effectiveness. Previous studies have found that colistin can synergy with various drugs; however, its exact mechanisms with hydrophobic drugs are still unrevealed. Generally, the membrane destruction of colistin is thought to be the essential trigger for its interactions with its partner drugs. Here, we use clustered regularly interspaced palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) for specifically mutating the binding site of one hydrophobic drug (flavomycin) and show that antimicrobial activity of flavomycin is critical for the synergy. Our results first give the evidence that the synergy is set off by colistin's membrane destruction and operated the final antimicrobial function by its partner drugs.

[Mechanism of mitochondrial dysfunction in polycystic ovary syndrome and traditional Chinese medicine intervention: a review].

Polycystic ovary syndrome(PCOS) is a highly prevalent endocrine and reproductive disorder characterized by ovulatory dysfunction, hyperandrogenism(HA), and polycystic ovarian morphology(PCOM). It is often accompanied by insulin resistance(IR), obesity, and metabolic disorders and can lead to cardiovascular diseases, endometrial carcinoma and many other late complications, seriously affecting the physical and mental health and quality of life in premenopausal women. The etiology of PCOS is still unknown and many scholars assume that mitochondrial dysfunction may represent a major pathogenic factor in PCOS in recent years. With a holistic view, treatment based on syndrome differentiation, and multi-system and multi-target treatment manner, traditional Chinese medicine(TCM) can mitigate the symptoms and signs of PCOS from multiple aspects. Although there have been reviews on the mechanism of mitochondrial dysfunction in PCOS, there is still a lack of reviews on the intervention of mitochondrial function by TCM to treat PCOS. Therefore, this paper focuses on the role of mitochondrial dysfunction in PCOS and summarizes the studies about the TCM intervention of PCOS by regulating the mitochondrial function, inflammation, oxidative stress(OS), autophagy, and apoptosis in the last five years, aiming to shed new light on the prevention and treatment of PCOS with TCM.

GRP78 protein metabolism in obese and diabetic rats: a study of its role in metabolic disorders.

OBJECTIVE: This study aimed to compare and analyze the expression and significance of the GRP78 protein in cochlear cell injury induced by a high glucose and high-fat diet in obese and diabetic rats. METHODS: Male SD rats were randomly divided into two groups: normal (NC) and high-fat (HF) groups. The NC group was fed a standard diet for eight weeks, while the HF group received a high-glucose, high-fat diet. The HF group was further categorized into the obesity group (OB group) and the type II diabetes mellitus group (T2DM group). To induce a type II diabetes mellitus (T2DM) model, the T2DM group received an intraperitoneal injection of a small dose of STZ (45 mg/kg). After four weeks on the original diet, body weight, blood glucose, blood lipid levels, and auditory brainstem response (ABR) thresholds were measured. The cochlea was dissected, and its morphology was observed using HE staining. Immunohistochemistry and western blotting were utilized to examine the expression level of the GRP78 protein in the cochlea. RESULTS: (1) The ABR threshold demonstrated a statistically significant difference between the T2DM group and the OB group (P < 0.05), as well as between the OB group and the NC group (P < 0.05). (2) Based on morphological comparisons from HE-stained sections, the T2DM group exhibited the most significant alterations in the number of cells in the spiral ganglion, the organ of Corti, and the stria vascularis of the cochlea. (3) The expression level of the GRP78 protein in the cochlea was higher in the T2DM group compared to the OB group (P < 0.05) and higher in the OB group compared to the NC group (P < 0.05). CONCLUSION: The findings indicate that the GRP78 protein plays a role in hearing loss caused by T2DM and hyperlipidemia. Moreover, T2DM is more likely than hyperlipidemia to be associated with hearing impairment.

Glioblastoma and Radiotherapy: a multi-center AI study for Survival Predictions from MRI (GRASP study).

BACKGROUND: The aim was to predict survival of glioblastoma at eight months after radiotherapy (a period allowing for completing a typical course of adjuvant temozolomide), by applying deep learning to the first brain MRI after radiotherapy completion. METHODS: Retrospective and prospective data were collected from 206 consecutive glioblastoma, IDH-wildtype patients diagnosed between March 2014-February 2022 across 11 UK centers. Models were trained on 158 retrospective patients from three centers. Holdout test sets were retrospective (n=19; internal validation), and prospective (n=29; external validation from eight distinct centers).Neural network branches for T2-weighted and contrast-enhanced T1-weighted inputs were concatenated to predict survival. A non-imaging branch (demographics/MGMT/treatment data) was also combined with the imaging model. We investigated the influence of individual MR sequences; non-imaging features; and weighted dense blocks pretrained for abnormality detection. RESULTS: The imaging model outperformed the non-imaging model in all test sets (area under the receiver-operating characteristic curve, AUC p=0.038) and performed similarly to a combined imaging/non-imaging model (p>0.05). Imaging, non-imaging, and combined models applied to amalgamated test sets gave AUCs of 0.93, 0.79, and 0.91. Initializing the imaging model with pretrained weights from 10,000s of brain MRIs improved performance considerably (amalgamated test sets without pretraining 0.64; p=0.003). CONCLUSIONS: A deep learning model using MRI images after radiotherapy, reliably and accurately determined survival of glioblastoma. The model serves as a prognostic biomarker identifying patients who will not survive beyond a typical course of adjuvant temozolomide, thereby stratifying patients into those who might require early second-line or clinical trial treatment.

Successful leadless pacemaker implantation in a patient with dextroversion of the heart: A case report.

BACKGROUND: Dextroversion is defined as the presence of dextrocardia with situs solitus, dextro-loop ventricles, and normally related great arteries. Dextrocardia can pose technical challenges when interventional treatments are required. However, the challenges posed by dextroversion can be amplified due to the disruption of typical anatomical relationships, the unpredictable positioning and boundaries of cardiac structures resulting from the shift, and the pathological processes influencing rotation. CASE SUMMARY: A 73-year-old woman with cardiac dextroversion suffered from a recurrence of atrial fibrillation after her radiofrequency catheter ablation and Despite the cessation of antiarrhythmic medications, there were episodes of sinus pauses and symptomatic bradycardia, with heart rates dropping as low as 28 beats per minute. CONCLUSION: Dextroversion makes the implantation of leadless pacemakers more challenging, and appropriate adjustments in fluoroscope angles may be crucial for intracardiac operations. Additionally, when advancing delivery systems, attention should be paid to rotational direction during valve-crossing procedures; changes in the perspective of posture angle between normal cardiac position and dextroversion can serve as references.

[Regional CT value in prediction of proximal femoral fracture].

OBJECTIVE: To investigate CT values of cancellous bone in femoral neck in adults over 60 years with proximal femoral fractures. METHODS: From January 2020 to December 2020, a retrospective analysis was performed on 280 subjects aged 60 years or older who underwent bilateral hip CT examination, including 85 males and 195 females, 120 on the left side and 160 on the right side, aged 75 (66, 82) years old. One hundred thirty-six patients with proximal femoral fractures were included in study group and 144 patients without fractures were included in control group. GEOptima CT was used to scan and reconstruct horizontal, coronal and sagittal layers of proximal femur. CT values of cancellous bone in femoral neck were measured and compared between two groups. The relationship between CT values of cancellous bone of femoral neck and proximal femoral fracture was analyzed statistically. RESULTS: In terms of age, fracture group aged 79(73.3, 85.0) years old, non-fracture group aged 69.5 (64.0, 78.8) years old, and had significant difference in age between two groups (P<0.05). In terms of CT value, regional CT value in fracture group was 8.62(-3.62, 27.15) HU, which was lower than that in non-fracture group 34.31(-5.93, 71.74) HU(P<0.05). CT value on coronal view in fracture group was -8.48(-30.96, 17.46) HU, which was lower than that in non-fracture group 40.49(5.55, 80.71) HU (P<0.05). CT value on sagittal view in fracture group was -31.28(-54.91, -5.11) HU, which was lower than that in non-fracture group 7.74(-20.12, 44.54) HU (P<0.05). CT values on horizontal view in fracture group was 0.17(-23.13, 24.60) HU, which was lower than that in non-fracture group 46.40(10.42, 85.18) HU(P<0.05). The mean regional CT values among three planes in the fracture group were lower than those in the non-fracture group. Logistic regression analysis showed coronal CT value was influencing factors of proximal femoral fracture, and it could be written into regression equations that predict probability of fracture. CONCLUSION: In adults aged over 60 years old, CT values of cancellous bone of femoral neck decreased with increasing age. The smaller CT value of cancellous bone of femoral neck, the greater risk of proximal femoral fracture.

Proteomic and metabolomic analysis of ageing beef exudate to determine that iron metabolism enhances muscle protein and lipid oxidation.

The study aimed to assess differences in proteomic and metabolite profiles in ageing (1, 2, 4, and 6 days at 4 °C) beef exudates and determine their relationship with beef muscle iron metabolism and oxidation. Proteomic and metabolomic analyses identified 877 metabolites and 1957 proteins. The joint analysis identified 24 differential metabolites (DMs) and 56 differentially expressed proteins (DEPs) involved in 15 shared pathways. Ferroptosis was identified as the only iron metabolic pathway, and 4 DMs (l-glutamic acid, arachidonic acid, glutathione and gamma-glutamylcysteine) and 5 DEPs (ferritin, phospholipid hydroperoxide glutathione peroxidase, heme oxygenase 1, major prion protein, and acyl-CoA synthetase long chain family member 4) were involved in iron metabolism by regulating heme and ferritin degradation, Fe2+ and Fe3+ conversion, arachidonic acid oxidation and inactivation of glutathione peroxidase (GPX) 4, leading to increased levels of free iron, ROS, protein and lipid oxidation (P < 0.05). Overall, abnormal iron metabolism during ageing induced oxidative stress in muscle tissue.

The anti-leukemia activity and mechanisms of shikonin: a mini review.

Leukemia encompasses a group of highly heterogeneous diseases that pose a serious threat to human health. The long-term outcome of patients with leukemia still needs to be improved and new effective therapeutic strategies continue to be an unmet clinical need. Shikonin (SHK) is a naphthoquinone derivative that shows multiple biological function includes anti-tumor, anti-inflammatory, and anti-allergic effects. Numerous studies have reported the anti-leukemia activity of SHK during the last 3 decades and there are studies showing that SHK is particularly effective towards various leukemia cells compared to solid tumors. In this review, we will discuss the anti-leukemia effect of SHK and summarize the underlying mechanisms. Therefore, SHK may be a promising agent to be developed as an anti-leukemia drug.

Impact of high-power short-duration atrial fibrillation ablation technique on the incidence of silent cerebral embolism: a prospective randomized controlled study.

BACKGROUND: High-power short-duration (HPSD) ablation strategy has emerged as a popular approach for treating atrial fibrillation (AF), with shorter ablation time. The utilized Smart Touch Surround Flow (STSF) catheter, with 56 holes around the electrode, lowers electrode-tissue temperature and thrombus risk. Thus, we conducted this prospective, randomized study to investigate if the HPSD strategy with STSF catheter in AF ablation procedures reduces the silent cerebral embolism (SCE) risk compared to the conventional approach with the Smart Touch (ST) catheter. METHODS: From June 2020 to September 2021, 100 AF patients were randomized 1:1 to the HPSD group using the STSF catheter (power set at 50 W) or the conventional group using the ST catheter (power set at 30 to 35 W). Pulmonary vein isolation was performed in all patients, with additional lesions at operator's discretion. High-resolution cerebral diffusion-weighted magnetic resonance imaging (hDWI) with slice thickness of 1 mm was performed before and 24-72 h after ablation. The incidence of new periprocedural SCE was defined as the primary outcome. Cognitive performance was assessed using the Montreal Cognitive Assessment (MoCA) test. RESULTS: All enrolled AF patients (median age 63, 60% male, 59% paroxysmal AF) underwent successful ablation. Post-procedural hDWI identified 106 lesions in 42 enrolled patients (42%), with 55 lesions in 22 patients (44%) in the HPSD group and 51 lesions in 20 patients (40%) in the conventional group (p = 0.685). No significant differences were observed between two groups regarding the average number of lesions (p = 0.751), maximum lesion diameter (p = 0.405), and total lesion volume per patient (p = 0.669). Persistent AF and CHA2DS2-VASc score were identified as SCE determinants during AF ablation procedure by multivariable regression analysis. No significant differences in MoCA scores were observed between patients with SCE and those without, both immediately post-procedure (p = 0.572) and at the 3-month follow-up (p = 0.743). CONCLUSIONS: Involving a small sample size of 100 AF patients, this study reveals a similar incidence of SCE in AF ablation procedures, comparing the HPSD strategy using the STSF catheter to the conventional approach with the ST catheter. TRIAL REGISTRATION: Clinicaltrials.gov: NCT04408716. AF = Atrial fibrillation, DWI = Diffusion-weighted magnetic resonance imaging, HPSD = High-power short-duration, ST = Smart Touch, STSF = Smart Touch Surround Flow.

[Effect of polishing method and time on the fitness of CAD/CAM zirconia ceramic crown].

PURPOSE: To compare the effect of different polishing methods and time treatment on the fitness of CAD/CAM zirconia ceramic crowns. METHODS: Sixteen intact maxillary premolars were randomly divided into two groups, group A was treated with silicon carbide burs, while group B was treated with tungsten steel burs. At different polishing time points of the same tooth, digital impressions of each group were obtained, which were used to manufacture CAD/CAM zirconium ceramic crowns. After trial fitting, the gap impressions were obtained by using silicone rubber replication method, and the marginal and internal discrepancies were assessed. The data were statistically analyzed with SPSS 21.0 software package. RESULTS: The difference between the gap values of the marginal and internal markers of group A and group B was not statistically significant(P＞0.05). Compared with the no-polishing process, the differences of the marginal gap (39.67±8.35) µm and internal gap (45.18±7.16) µm of group A polished for 4 min, and the marginal gap (51.25±14.73) µm, and internal gap (48.56±6.45) µm of group B polished for 3 min, as well as the marginal gap (48.87±8.90) µm, and internal gap (45.99±7.12) µm of group B polished for 4 min, were all significant(P＜0.05). CONCLUSIONS: CAD/CAM zirconia ceramic crowns treated with silicon carbide bur for polishing 4 min and tungsten steel for 3 min has the best fitness.

Prevalence, risk factors and multiple outcomes of treatment delay in Chinese patients with schizophrenia.

BACKGROUND: People with schizophrenia often delay treatment. This issue is not fully understood, particularly in low-and middle-income countries. This study aimed to elucidate the prevalence, risk factors and multiple outcomes of treatment delay in schizophrenia in a Chinese metropolis. METHODS: A two-stage whole cluster sampling survey was conducted in Beijing, China in 2020. A total of 1,619 patients with schizophrenia were included. Heterogeneity between groups and the changing trend of treatment delay were presented. Regression modelling methods were used to examine both the risk factors for treatment delay and related outcomes at individual and family levels. RESULTS: The median treatment delay for schizophrenia was 89 days (about 13 weeks). 49.35% surveyed patients delayed treatment for more than three months. Early age of onset, low level of education, living in well developed districts were important risk factors. Treatment delay in schizophrenia was significantly associated with patients' poor medication adherence, comorbidity status and poor social functioning. It also increased the negative impact of the illness on families. CONCLUSIONS: This study accumulated evidence of treatment delay in schizophrenia in China. It occurs even in the metropolis where mental health resources are relatively adequate. Further targeted interventions to raise public awareness should be crucial to reduce treatment delay.

Abnormal cortical surface-based spontaneous and functional connectivity in the whole brain in lifelong premature ejaculation patients.

Recent research has highlighted structural and functional abnormalities in the cerebral cortex of patients with premature ejaculation (PE). These anomalies could play a pivotal role in the physiological mechanisms underlying PE. This study leveraged functional magnetic resonance imaging (fMRI), a noninvasive technique, to explore these neural mechanisms. We conducted resting-state fMRI scans on 36 PE patients and 22 healthy controls (HC), and collected data on Premature Ejaculation Diagnostic Tool (PEDT) scores and intravaginal ejaculation latency time (IELT). Employing a surface-based regional homogeneity (ReHo) approach, we analyzed local neural synchronous spontaneous activity, diverging from previous studies that utilized a volume-based ReHo method. Areas with significant ReHo differences between PE and HC groups underwent surface-based functional connectivity (FC) analysis. Significant discrepancies in ReHo and FC across the cortical surface were observed in the PE cohort. Notably, PE patients exhibited decreased ReHo in the left triangular inferior frontal gyrus and enhanced ReHo in the right middle frontal gyrus. The latter showed heightened connectivity with the left lingual gyrus and the right orbital superior frontal gyrus. Furthermore, a correlation between ReHo and FC values with PEDT scores and IELT was found in the PE group. Our findings, derived from surface-based fMRI data, underscore specific brain regions linked to the neurobiological underpinnings of PE.

Hypusination-induced DHPS/eIF5A pathway as a new therapeutic strategy for human diseases: A mechanistic review and structural classification of DHPS inhibitors.

The discovery of new therapeutic strategies for diseases is essential for drug research. Deoxyhypusine synthase (DHPS) is a critical enzyme that modifies the conversion of the eukaryotic translation initiation factor 5A (eIF5A) precursor into physiologically active eIF5A (eIF5A-Hyp). Recent studies have revealed that the hypusine modifying of DHPS on eIF5A has an essential regulatory role in human diseases. The hypusination-induced DHPS/eIF5A pathway has been shown to play an essential role in various cancers, and it could regulate immune-related diseases, glucose metabolism-related diseases, neurological-related diseases, and aging. In addition, DHPS has a more defined substrate and a well-defined structure within the active pocket than eIF5A. More and more researchers are focusing on the prospect of advanced development of DHPS inhibitors. This review summarizes the regulatory mechanisms of the hypusination-induced DHPS/eIF5A pathway in a variety of diseases in addition to the inhibitors related to this pathway; it highlights and analyzes the structural features and mechanisms of action of DHPS inhibitors and expands the prospects of future drug development using DHPS as an anticancer target.

Surgical Treatment of Cervical Kyphosis and Atlantoaxial Dislocation in a Child With Loeys-Dietz Syndrome: A Case Report and Literature Review.

We report the case of a 3-year-old child with Loeys-Dietz syndrome, a rare genetic connective tissue disorder. The young girl had concurrent cervical kyphosis, atlantoaxial dislocation (AAD), and spinal cord compression. Posterior occipitocervical fusion was performed. Postoperative examination and clinical manifestations confirmed that all pedicle screws were satisfactorily placed, cervical kyphosis and AAD were corrected, and spinal cord compression was relieved. At the 1-year postoperative follow-up, the patient had recovered well, indicating that our operation was successful. To the best of our knowledge, this is the first reported surgical case of cervical kyphosis and AAD caused by Loeys-Dietz syndrome.

Premature ovarian insufficiency: a review on the role of oxidative stress and the application of antioxidants.

Normal levels of reactive oxygen species (ROS) play an important role in regulating follicular growth, angiogenesis and sex hormone synthesis in ovarian tissue. When the balance between ROS and antioxidants is disrupted, however, it can cause serious consequences of oxidative stress (OS), and the quantity and quality of oocytes will decline. Therefore, this review discusses the interrelationship between OS and premature ovarian insufficiency (POI), the potential mechanisms and the methods by which antioxidants can improve POI through controlling the level of OS. We found that OS can mediate changes in genetic materials, signal pathways, transcription factors and ovarian microenvironment, resulting in abnormal apoptosis of ovarian granulosa cells (GCs) and abnormal meiosis as well as decreased mitochondrial Deoxyribonucleic Acid(mtDNA) and other changes, thus accelerating the process of ovarian aging. However, antioxidants, mesenchymal stem cells (MSCs), biological enzymes and other antioxidants can delay the disease process of POI by reducing the ROS level in vivo.

Tf2O/DMSO-mediated dual activation of aryl phosphinate to access various aryl phosphonates.

A metal-free method for the dual activation of aryl phosphinate has been developed; the P-H and P-O bonds are sequentially activated by the Tf2O/DMSO system. Without the requirement of metals and unstable P-reagents, this one-pot procedure provides a convenient and practical access to a variety of aryl phosphonates. A mechanism involving twice generation of electrophilic P-species and two SN-processes is proposed on the basis of the control experiments.