Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O2–• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin’s effects on glutamate-evoked O2–• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca2+]i and [O2–•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O2–• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O2 consumption rate (OCR). Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca The present study aims to investigate the effect of insulin on glutamate-induced O2–• generation in cultures of rat cortical neurons, with an emphasis on the relationship between the dynamics of superoxide production and the increase in [Ca2+]i in single neurons in response to the application of exogenous Glu i and [O2–•], wherein intracellular [O2–•] includes both cytosol and mitochondrial superoxide. To investigate whether insulin can influence glutamate-evoked changes in intracellular Ca2+ and superoxide, neurons were treated with Glu (100 μM) in the presence or absence of insulin (100 nM), and the intracellular [O2–•] and [Ca2+]i dynamics were monitored for 30 min  and [O2–•] . Tukey’s post-test showed that Glu-treated neurons had significantly higher levels of [Ca2+]i (p < 0.0001) and superoxide (p < 0.001 to p < 0.0001) compared to those of intact neurons. At 30 min after the addition of Glu, the mean levels of [O2−•] were significantly higher (p < 0.0001) compared to those in the intact control neurons. Insulin significantly decreased the Glu-evoked [Ca2+]i (p < 0.01 to p < 0.0001) and [O2–•] (p < 0.05 to p < 0.0001). At 30 min, superoxide production in neurons treated with insulin and Glu was 35% lower (p < 0.0001) than in neurons treated with glutamate alone. Primary cultured rat cortical neurons were coloaded with the low affinity fluorescent Car 30 min . Two-way2+]i and [O2–•], indicating a major role for NMDA receptors (p < 0.0001). There were no statistically significant differences in [Ca2+]i or [O2–•] between neurons treated with MK 801 and glutamate in the presence (p > 0.05) or absence of insulin (p > 0.05). These results suggest that NMDAR-gated Ca2+ influx is critically involved in the glutamate-evoked superoxide generation of superoxide in cortical neurons exposed to glutamate, regardless of the presence or absence of insulin. MK 801, a non-competitive NMDAR inhibitor, completely abolished Glu-induced increases in [Ca2+]i and intracellular superoxide levelsin neurons treated with glutamate in the absence of insulin (p < 0.0001) and in the presence of insulin  within a 30 min period of exposure to Glu. A significant Pearson linear correlation was found between the mean levels of [Ca2+]i and mitochondrial superoxide, respectively, and then treated with 100 μM glutamate in the presence or absence of 100 nM insulin . Rat cortical neurons were coloaded with Fura-FF and Mito-HE to simultaneously monitor changes in intracellular [Ca insulin . 2+]i  and mitochondrial superoxide levels . Bonferroni’s post-test shows that insulin significantly diminished glutamate-evoked rise in [Ca2+]i (p < 0.05) and mitochondrial superoxide levels (p < 0.01 to p < 0.0001). At 30 min after treatment, the mean superoxide level in neurons treated with insulin and Glu was lower by 49% (p < 0.01) compared to neurons treated with Glu alone. There was a significant linear correlation between [Ca2+]i and superoxide levels for Glu-treated neurons in the absence of insulin (p < 0.0001) and in the presence of insulin . Two-way ANOVA with repeated measures revealed significant effects of time and insulin on glutamate-induced changes in both [Ca2+]i in neurons. Thus, taken together, these results suggest that insulin prevents the glutamate-induced increase both in intracellular and mitochondrial superoxide production by diminishing the Glu-dependent rise in [Ca2–•] generation and oxygen metabolism during excitotoxicity of Glu, we measured oxygen consumption rates (OCR) in cortical neurons exposed to Glu (100 µM) in the presence or absence of insulin (100 nM). Kruskal–Wallis, followed by Dunn’s multiple comparison test, revealed that Glu exposure reduced maximal respiration by 31% (p < 0.001) and spare respiratory capacity by 58% (p < 0.001) compared to control. In agreement with the data shown in 2+]i and [O2–•] levels are concomitant with the glutamate-induced decrease in maximal respiration and SRC. Insulin significantly attenuated the inhibitory effects of Glu on maximal respiration (p < 0.01) and spare respiratory capacity (p < 0.05) compared to Glu alone. Since the latter relates to the amount of extra adenosine triphosphate (ATP) that can be produced by oxidative phosphorylation in response to increased energy demand, the protective effects of insulin against a glutamate-evoked increase in [Ca2+]i and superoxide levels may be related to its preventive action against glutamate-induced impairment of mitochondrial bioenergetics, given that Ca2+, ATP, and superoxide exist in a network, with each able to control the others i and preventing the onset of DCD [2–•], the main sources of excitotoxic ROS in neurons , the inhibitory effect of insulin on [O2–•] accumulation appears to be related to its effect on [Ca2+]i dynamics.Correlations close to linear were observed between mean i and [O2–•], regardless of the presence or absence of insulin, indicating a pivotal role for NMDA-type ionotropic glutamate receptors (2+]i and [O2–•] showed that the growth of [O2–•] occurs synchronously with the onset of DCD in each cell. Cessation of Ca2+ entry into the cell with the help of MK-801 abolished the formation of [O2–•] (2+ in the buffer (not shown). These results are consistent with previous data on the key role of Ca2+ influx through NMDAR in ROS accumulation during glutamate excitotoxicity.MK-801, an inhibitor of NMDAR-dependent Caeceptors . Simultaf [O2–•] . Similar2+ influx . However, a contribution of mitochondrial superoxide production to total [O2–•] production cannot be quantified from Mito-HE and HE fluorescence data, as these probes have different rates of oxidation with superoxide i. Before DCD development, NMDAR-gated Ca2+ influx is counterbalanced with mitochondrial Ca2+ uptake and its efflux produced by the plasma membrane Ca2+-ATPase and the Na+/Ca2+-exchanger (NCX). The development of DCD and profound mitochondrial depolarization cancel Ca2+ uptake by mitochondria and reverse F1Fo-ATP synthase to glycolytic ATP-consuming ATPase i i in excitotoxicity. NCX  plays a for Ca2+ ,37. NCX- [Ca2+]i ,29,38,39the cell . Therefo2+]i and superoxide levels concomitantly with the decrease in SRC  . Insuline in SRC . Insulinepletion , where S2+]i, an increase in [ATP]i, and a decrease in mitochondrial and cytosolic superoxide formation. Short-term insulin exposure in our experiments appears to be relevant for in vivo conditions of intranasal insulin therapy. Intranasally managed insulin has been shown to reach a peak value in the rat brain 15 min after administration . The rap2+]i and (ii) decreasing the superoxide yield rate by the same increment in [Ca2+]i. The effects of insulin on glutamate excitotoxicity are associated with improved spare respiratory capacity and the ability to produce additional ATP due to oxidative phosphorylation. Given that generation of [O2–•] generation is considered to be causal for neuronal death, the prevention of the onset of oxidative stress with insulin appears to at least partially explain its neuroprotective action in glutamate excitotoxicity. We found that short-term exposure to insulin prevents glutamate-evoked superoxide generation in cultures of rat cortical neurons by diminishing the glutamate-induced rise in [Ca2+]i, was on average 32% and 46% lower in insulin-treated neurons, estimated using HE and Mito-HE. This suggests that the increase of [Ca2+]i, while necessary, is not the only factor affecting the magnitude of superoxide production. Given a recent finding that NMDAR-induced superoxide production requires non-ionotropic NMDAR signaling via PI3K, the inhibitory effect that insulin has on superoxide generation may perhaps be related to interference between insulin receptor i, cortical neurons were loaded with a low-affinity Ca2+ indicator, Fura-FF, in the form of acetoxymethyl ester  in the presence of a non-ionic detergent, Pluronic F-127 (0.02%) which was acquired from Molecular Probes  to facilitate the penetration of Fura-FF into the cells. We used the low-affinity calcium dye Fura-FF in the experiments because long-term exposure to high doses of Glu (100 µM) results in such a high intracellular calcium concentration ([Ca2+]i) that high-affinity probes, such as Fura-2/AM, are rapidly saturated with calcium and does not reflect true changes in [Ca2+]i [2+]i, including in experiments on Glu excitotoxicity [2+]i in different phases of OCD and presented corresponding images proving that Fura-FF AM penetrates well into the cell and is fairly evenly distributed in the cytosol. Mitochondria in cells (in neurons in particular) are able to accumulate Fura-FF only when using concentrations of Fura-FF/AM that are 2 orders of magnitude higher than those that are usually used to load cells. Therefore, mitochondria can only be loaded in a state isolated from cells.To measure [Ca [Ca2+]i ,35. Furas of Glu 0 µM resutoxicity ,29. Comptoxicity . In our s of Glu 0 µM resu2+]i occur in the cytosol, and Fura-2, which is widely used in research, is quickly saturated with calcium and does not reflect true changes in [Ca2+]i in the cytosol [2+]i and [O2–•], cells were loaded with 1 μM of dihydroethidium (HE) fluorescent probe or triphenyl phosphonium linked hydroethidine (Mito-HE) fluorescent probe, a.k.a. MitoSOX Red, for the last 20 min of the “Fura-FF/AM loading period” in buffer at 37 °C. After loading, the cells were washed with saline of the following composition (in mM): NaCl—135; KCl—5; CaCl2—2; MgCl2—1; HEPES—20; glucose—5, pH 7.4. Insulin, at 100 nM, was added 5 min before 100 μM glutamate in Mg2+ free. To wash out Glu, a nominally Ca2+-free solution was used to exclude any pathways for Ca2+ entry from the buffer into the cytosol in the post-glutamate period. To assess the amount of Ca2+ accumulated by mitochondria, mitochondria in the final part of the experiments were depolarized using the FCCP protonophore (1 μM). To calibrate the maximum signal of the Ca2+ indicator, ionomycin  in the presence of 5 mM Ca2+ was added at the end of the experiment. The solutions were changed 3 times, 1 mL each. The cells were incubated on a microscope stage at 24–25 °C . The Olympus IX-71 microscope was equipped with a 20x fluorite objective for measuring the fluorescence of various fluorescent probes. The light source is a Xenon arc lamp (175 W), the light of which alternately passes through interference filters installed in a computer-controlled wheel, which provides filter change in 50–200 ms . A shutter between the lamp and the wheel with light filters block the radiation for the time when the signal is not recorded, preventing photo destruction of the object under study. The dichroic mirror reflects the exciting light toward the objective and transmits the radiation from the sample (colored cell) toward the detector. A lens focuses the excitation light on the sample and collects its fluorescence by directing it through a dichroic mirror to a detector. Cell fluorescence passed through the emission filter wheel, which was controlled synchronously with the excitation wheel. The image was detected using a CCD camera CoolSNAP HQ2 . The image analysis system was controlled with the MetaFluor 6.2 computer program . The recording signals from cell indicators and images were recorded every 30 s. Fura-FF fluorescence was excited alternately at 340 and 380 nm and recorded at 525 nm. HE and Mito-HE excitation/emission fluorescence were 565/610 nm. Cell fluorescence was normalized relative to basal levels in resting cells; the typical interval between the acquisition of successive images (time-lapse) was 30 s.Fura-FF was tested on neurons precisely because under the action of Glu, such high i. Given that superoxide causes neuron death, these results seem to explain the neuroprotective action of insulin in glutamate excitotoxicity. Insulin effects are also related to the improvement of spare respiratory capacity, the ability to produce additional ATP by oxidative phosphorylation in neurons during glutamate action.We found that insulin prevents glutamate-evoked intracellular and mitochondrial superoxides generation in primary cultures of rat cortical neurons by decreasing the glutamate-induced rise in [Ca


Lacticaseibacillus rhamnosus JB-1, and Limosilactobacillus reuteri 6475 treatment and performed Gene Set Enrichment Analysis (GSEA) to identify enrichment in pathway activity. L. rhamnosus, but not L. reuteri treatment altered several pathways in the blood and hippocampus, and the rhamnosus could be clearly distinguished based on mRNA profile. In particular, L. rhamnosus treatment modulated the activity of interferon signaling, JAK/STAT, and TNF-alpha via NF-KB pathways. Our results highlight that psychobiotics can induce complex changes in host gene expression, andin understanding these changes, we may help fine-tune selection of psychobiotics for treating mood disorders.Discovery of the microbiota-gut–brain axis has led to proposed microbe-based therapeutic strategies in mental health, including the use of mood-altering bacterial species, termed psychobiotics. However, we still have limited understanding of the key signaling pathways engaged by specific organisms in modulating brain function, and evidence suggests that bacteria with broadly similar neuroactive and immunomodulatory actions can drive different behavioral outcomes. We sought to identify pathways distinguishing two psychoactive bacterial strains that seemingly engage similar gut–brain signaling pathways but have distinct effects on behaviour. We used RNAseq to identify mRNAs differentially expressed in the blood and hippocampus of mice following  Anxiety and depression are two of the most common mood disorders in the western world becoming increasingly prevalent in millennials and adolescents ,2. The CLacticaseibacillus rhamnosus JB-1, which has previously been shown to reduce anxiety and depression-like behaviours in mice [Numerous direct and indirect interactions between bacteria endemic to the gut, and the central nervous system characterize what is known as the microbiota-gut–brain axis . The int in mice ,9,10.L. rhamnosus JB-1.It is incompletely understood how JB-1 facilitates these cognitive and behavioural changes, although both the peripheral nervous system and the immune system are critical mediators ,9,11. SpLimosilactobacillus reuteri 6475 (LR6475) is a probiotic bacteria that has previously been shown to modulate social behaviours; rescuing autism-spectrum-disorder-like social deficits induced by a maternal high-fat diet in mice [ in mice . Mechani in mice ,18. LR64 in mice ,20. Howe in mice . The reaHere, in an attempt to identify potential pathways distinguishing two psychoactive bacterial strains that seemingly engage similar gut–brain signaling pathways but have distinct effects on behaviour, we compare transcriptomic changes in blood and hippocampus, a region of the brain responsible for memory and emotion and closely linked with depression ,23,24, fp-value = 0.049)  of normalized, filtered mRNA in the blood of mice shows no distinct groups between PBS and LR6475 fed mice; however, JB-1 fed mice differ greatly from the cluster PBS and LR6475-fed mice form along both PC 1 and 2 (adonis = 0.049) A. As the= 0.049) B and a f= 0.049) D; howeveTo elucidate additional sources of grouping along PC1 and 2 in the blood, Gene Set Enrichment Analysis was performed, comparing KEGG and Hallmark gene set expression in JB-1-fed mice to LR6475 and to PBS-fed independently. LR6475 and PBS-fed mice were not compared as their groups were not distinct in PCA. Many pathways from both KEGG and Hallmark were found differentially expressed in both directions, in both JB-1 comparisons, and are summarized in . All of p-value = 0.17)  A. Only 2 = 0.17) B. When c = 0.17) C,D; howep-value = 1.3 × 10−4) and LR6475-fed mice  compared to PBS-fed mice, and KEGG_VIBRO_CHOLERAE_INFECTION was enriched in PBS compared to LR6475 . With so few discerning features between individual gene expression and enrichment analysis, it remains unclear what is driving the distinction seen between PBS and the other treatment groups in the PCA. Additional PCs were checked (up to PC12) but none alone explain the distinction.Enrichment analysis of hippocampal mRNA following treatment was also poorly able to identify the source of PCA group distinction. No Hallmark pathways were found differentially expressed in any of the three comparisons. KEGG_RIBOSOME was enriched in the hippocampi of both JB-1  which relies on unsupervised clustering of genes to construct a network with modules of commonly co-expressed genes.Using a power of 13 for blood and merging at a threshold of 0.05, 24 eigengenes were identified and mapped to the cluster dendogram A. There For hippocampal mRNA, a power of 30 was used, and merging at a threshold of 0.04, 9 eigengenes were identified and mapped to a cluster dendrogram A. No staHere, we examined blood and hippocampal transcriptional changes induced by two lactobacillus species that have previously been demonstrated to have distinct effects on behaviour in mice ,10,16,21Immunomodulatory actions have been described for both JB-1 and LR6475 and in the case of JB-1 these have been demonstrated to mediate effects on behaviour ,9,10,16.We also observed marked changes in mRNA related interferon signaling with significant diminution of HALLMARK_INTERFERON_ALPHA_RESPONSE, HALLMARK_INTERFERON_GAMMA_RESPONSE, and HALLMARK_INFLAMMATORY_RESPONSE in the blood of mice treated with JB-1 in comparison to both PBS and reuteri 6475. The Interferons are known to play a role in the link between the immune system and mood disorders. INF-α is used to treat hepatitis C and is associated with a 30–70% increased risk of emergent depression . InterfeHALLMARK_TNFA_SIGNALLING_VIA_NFKB was underexpressed in both JB1 blood comparisons. These are genes that are regulated by NF-KB in response to TNF-α. TNF-α signaling through NF-KB has previously been shown to activate microglia and increase neuroinflammation in mice showing depression-like behaviour . Mice inFinally, FKBP12, one of the genes increased in expression in the blood of JB-1-fed animals compared to the other treatment groups, is a known inhibitor of mTOR signaling . This maThe Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway controls several processes in the cerebral cortex and hippocampus, including microglial activation, synaptic plasticity, gliogenesis, and neurogenesis ,46,47. HWithin all comparisons made, only two genes were significantly overexpressed in the hippocampus-TPPP3 and SGK1 which were altered in the JB-1 vs. PBS-fed comparison. Tubulin polymerization-promoting protein family member 3 (TPPP3) is a protein that regulates microtubule dynamics , and SerSeveral mRNAs were upregulated in the blood of JB1-fed mice compared to both LR6475 and PBS-fed mice. They were: FKBP1A, SNRPN, CELF4, GPM6B, APBB1, NCDN, RUNDC3A, CPE, and CLSTN1.Neuronal membrane glycoprotein gene (GPM6B) codes for a protein involved in bone formation and osteoblast function, as well as being a binding partner of the serotonin transporter (SERT) ,59. It hCarboxypeptidase E (CPE) codes for an exopeptidase that removes C-terminal lysine or arginine acids from peptides . Rodrigun = 118) compared to healthy subjects (n = 236) [Calsyntenin-1 (CLSTN1) codes for a protein that encourages vesicle association with KLC1 in axonal anterograde transport . CLSTN1 n = 236) . The mecn = 236)  confirmen = 236)  were colLacticaseibacillus rhamnosus JB-1 (2 × 10⁹), Limosilactobacillus reuteri 6475 (2 × 10⁹), or PBS . Mice were gavaged once per day for 2 weeks and sacrificed 3 h after the last gavage. Trunk blood was collected and whole brains were flash frozen and stored. Later, hippocampi of alternating side half brains were isolated and stored for RNA isolation.7–9-week-old male balb/c mice from Charles River Laboratories were orally gavaged with 200 µL of either Total RNA was isolated from fresh whole blood using a PureLink RNA mini kit for total RNA isolation and using the manufacturer-recommended protocol for whole blood extraction.Total RNA from hippocampi was isolated first by homogenizing the tissue with mortar and pestle in lysis buffer, followed by up-down pipetting through a 27-gauge syringe. After the tissue was completely homogenized, the same PureLink RNA mini kit was used to extract total RNA using the manufacturer-recommended protocol for tissue extraction.Four samples from each gavage group, totaling at 12 samples for blood and hippocampus, underwent paired-end RNAseq  on an illumina NextSeq for mRNA discovery and analysis.p-value < 0.05, and an |FC| > 1.5. p-values were adjusted using Benjamini–Hochberg method [Raw RNAseq data were adaptor trimmed and aligned using the ‘RNA-Seq Alignment’ app in Illumina BaseSpace which uses the Spliced Transcripts Alignment to a Reference (STAR) alignment method with the USCS mm10 refseq gene annotation file . Next trg method .Percent variables and principal components were calculated using DESeq2′s plotPCA function on the list of internally normalized and filtered genes, and then graphed using ggplot2 .p-values values generated by DESeq2′s ‘results’ function were used by the generally applicable gage  package in R to generate enrichment results to the KEGG and Hallmark pathway gene sets [p-value < 0.05.Fold change and adjusted ene sets . KEGG paene sets  and showHere, we identified several pathways and genes that may be associated with JB-1, treatment that may plausibly be related to the effects of these organisms on behavior, summarized in . Some arThere are certain limitations to the current study. The brains used in the study were not perfused following collection, and therefore could contain a small amount of blood from within capillaries in the hippocampus. Furthermore, only male mice were used, there is evidence that the outcome of gut–brain signaling can be sex dependent and thus sex comparisons would be worthy of examination in the future. This is particularly pertinent as in humans mental health disorders disproportionately affect women. Finally, WGCNA recommends 15 samples minimum for analysis and we performed the analysis with 12, which may have reduced the precision of the results.Overall, our results highlight that microbes labeled as psychobiotics, or potential psychobiotics, induce complex changes in systemic gene expression which are far from uniform between organisms. A better understanding of the many pathways impacted by individual organisms may help develop more tailored microbe-based approaches to specific mental health issues.


Patient satisfaction is an important quality indicator of health care service. The concept of home‐based palliative care has been recently introduced in Bangladesh, but the patients' satisfaction with this care remained unexplored. This study aimed to assess the satisfaction of the cancer patients receiving this care.This cross‐sectional study was conducted among 51 surviving cancer patients above 18 years of age registered under the home‐based care service of the Department of Palliative Medicine, Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh. Data were collected by face‐to‐face interviews using a structured questionnaire based on the FAMCARE P16 questionnaire from February to March 2019. Descriptive analysis was done for the sociodemographic and satisfaction‐related indicators. A correlation matrix was done to see the correlation among the satisfaction indicators.R = 0.814, p < 0.001). Also, satisfaction regarding the provision of information and support provided to the family is highly correlated .The majority of the patients (88.2%) were satisfied with the service provided by the home care team. Most (76.5%) of the patients were women, and the mean age was 56.25 ± 14.8 years. The median duration of getting home‐based care was 4 months. Main satisfaction indicators were—assessment of physical symptoms (70.6%), providing information about pain management (70.6%), the inclusion of the family in decision making (76.5%), coordination of care between the members of the home care team (84.3%) and availability of doctors, nurses and palliative care assistants (74.5%). A high correlation was observed between satisfaction regarding the care of physical symptoms and provision of information (Despite the limitations, the overall satisfaction level of the patients regarding home‐based palliative care services in Bangladesh is very high. Home‐based palliative can be a solution to provide palliative care to patients who are unable to access institution‐based care and improve their quality of life. In Bangladesh, there are some isolated initiatives of providing home‐based palliative care have been taken. Approximately 0.6 million patients need palliative care in Bangladesh, but less than 4000 people have received this care until now.Patient satisfaction is one of the main indicators in assessing the quality of the service provided by the home‐care team. However, there is no such study done to assess the satisfaction level of cancer patients receiving this service. This study assessed the satisfaction with care in the cancer patients receiving home‐based palliative care in Bangladesh.22.1This cross‐sectional study was conducted amongst all surviving cancer patients currently registered under the Department of Palliative Medicine, BSMMU, Shahbag, Dhaka, Data collection was carried out in February and March 2019.2.2The home‐based palliative care service is provided by the Department of Palliative Medicine, BSMMU to the registered patients living within 20 km radius of the university. The home care team consists of one doctor, two to three nurses, and five trained palliative care assistants (PCAs). They work 5 days per week. Patients who are unable to visit hospital due to physical condition, distance, financial constraints, or lack of caregivers are considered to be eligible for this service irrespective of their disease stage. This service includes wound care, adjusting medications, general physical and mental care and follow‐up, interaction with the caregivers to give temporary respite from their caregiving duties, as well as bereavement support. The PCA includes specially trained individuals who do the initial visits. They are involved in minor wound care, general physical care, helping the family caregivers, and listening to the patients' and their caregivers' problems. Doctors and nurses visit each patient once a week. They are involved in major wound care, adjustment of medication, and communication with the patients and their family members about which the PCAs cannot elaborate. The visits are need based. Usually, every patient gets two to three visits per month, although extra visits are given based on patients' condition and caregiver demand.Satisfaction is the fulfillment of the individual needs and expectations of the patients and their families, and is assessed by a two‐way communication between the receivers of the care and the provider of the care.2.3All the surviving cancer patients who registered under this service up to February 2019, above 18 years of age, willing to participate, and got at least three visits from the home care team were included in the study. Those who were delirious, disoriented, or unable to communicate were excluded. Those caregivers (paid or family members) who take care of the patients at least 5 days per week are included in the study. Occasional caregivers were excluded.2.4According to the Center of Palliative Care (CPC) database up to February 2019, the number of registered cancer patients receiving home‐based palliative care was 60. During data collection three patients died, four patients were not eligible for the study due to delirium, and three patients refused to give informed consent. So the final sample size of the study was 51.2.5The questionnaire had two major parts. The first part contained the sociodemographic, disease, treatment, and primary caregiver‐related information collected from the hospital record.The second part of the questionnaire contained questions from the FAMCARE P‐16 questionnaire developed by Lo et al.2.6Initial data were collected from the hospital case sheets .The investigators accompanied the home care team to the patients' home, and the interviews were conducted in their presence. Informed consent was obtained from both the patients and their primary caregivers. Mini Mental State Examination (MMSE) was done to determine the consent‐giving capacity of the patient. The consent was obtained either in written or verbal form depending on the patients' condition.The patients and the caregivers were recruited in pair and were interviewed together. The duration of each interview was 30 minutes to 1 hour. Very frail patients were given multiple visits to complete an interview.2.7All data were analyzed by SPSS version 22.0 after editing and logical checking.Categorical variables such as sex, education, marital and occupational status, knowledge and belief about the disease prognosis, treatment, and side effects, the relationship of the primary caregiver with the patient were reported as frequency and percentage. Continuous variables such as age, monthly family income, duration of getting home‐based palliative care were presented in mean, SD, and median as appropriate. Number of satisfied patients with each component was presented in frequency and percentage.The level of satisfaction was presented in three categories based on mean and SD. The value below lower limit of mean − 1  SD was categorized as not satisfied, the range between upper and lower limit of mean ± 1 SD was categorized as satisfied and the value above mean + 1 SD was categorized as very satisfied.p < 0.05 was considered as significant.Correlation matrix was done to see the correlation among the satisfaction indicators. 3The majority (76.5%) of the patients were women, and the mean age was 56.25 ± 14.8 years. More than half (58.8%) of the patients were married and lived with their partners. Almost 97% of the patients had family members as their primary caregivers, mostly their children (53.2%) or spouses (29.8%), and 57.6% of the primary caregivers were women. Common sites of the primary cancer were breast (39.2%), genitourinary system (23.5%), and gastrointestinal tract (17.6%). More than half (55.8%) of the patients had metastasis at the time of referral to palliative care, and 80% of them were currently only on palliative management. The median duration of receiving home‐based palliative care of the patients was four months (ranging from 6 days to 1 year) , providing information about pain management (70.6%), the inclusion of the family in decision making (76.5%), coordination of care between the members the home care team (84.3%) and availability of doctors, nurses and PCAs in the time of need (74.5%) Table .The majority of the patients (88.2%) were satisfied with the service provided by the home care team Figure .R = 0.814, p < 0.001), followed by satisfaction regarding the support given to the family . Also, satisfaction regarding the provision of information and satisfaction regarding the support given to the family were highly and significantly correlated   Table .4Home‐based palliative care has been introduced recently in Bangladesh. This is the first study in Bangladesh assessing the satisfaction with the care of the patients' receiving such care.Measuring patients' satisfaction is important for evaluating the outcome of the care provided by the home care team. It gives valuable information about the patient's experience with the service, measures their compliance with the treatment, identifies the underlying weaknesses, and evaluates the performance of the home care team.The majority (80.4%) of the patients in this study are very satisfied with the attention given by the home care team members during the assessment of their sufferings. It indicates better communication between the home care team and the patients, where more than half (61%) of the patients attending government hospitals of Bangladesh show dissatisfaction about the health care providers' lack of attention to their symptom description.The majority of the patients in our study are very satisfied with the inclusion of family members in decision making (76.5%) and availability of the doctors and nurses in need (75.5%), in contrast to earlier studies where the main dissatisfaction arises from the long waiting time for the doctors and nurses in hospitals.One major limitation of this study is that this study was conducted on the home‐based palliative care service provided by a single institution, so the findings of the study cannot be generalized. Also the greater picture of home‐based palliative care in Bangladesh is not reflected in this study. Due to the cross‐sectional nature of this study, the perception of satisfaction among the patients cannot be measured over time.5Patient satisfaction is one of the important quality indicators of health care service. Despite the limitations, overall satisfaction on the care provided by the home care team is high. As home‐based palliative care continues to develop, further research is needed to evaluate the satisfaction level of the patients over time.Jheelam Biswas: Conceptualization; data curation; formal analysis; investigation; methodology; project administration; resources; software; validation; visualization; writing–original draft; writing–review and editing. Mithila Faruque: Conceptualization; formal analysis; project administration; software; supervision; validation; writing–original draft; writing–review and editing. Palash Chandra Banik: Conceptualization; data curation; formal analysis; methodology; project administration; software; supervision; validation; writing–original draft; writing–review and editing. Nezamuddin Ahmad: Funding acquisition; resources; supervision; validation; writing–original draft; writing–review and editing. Saidur Rahman Mashreky: Conceptualization; formal analysis; methodology; supervision; validation; visualization; writing–original draft; writing–review and editing.The authors declare no conflict of interest.Ethical approval for both the research and consent procedure  was obtained from the Ethical Review Committee, Bangladesh University of Health Sciences (BUHS), and permission for data collection was obtained from the Department of Palliative Medicine, BSMMU. The written informed consent was taken from all the eligible patients and their primary caregivers. As they were terminally ill patients, their health conditions were considered during data collection.The lead author Jheelam Biswas affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


In 10% of term deliveries and 40% of preterm deliveries, the fetal membrane (FM) ruptures before labor. However, the ability to predict these cases of premature rupture of membranes (PROM) and preterm premature rupture of membranes (PPROM) is very limited. In this paper, our objective was to determine whether a prediction method based on T2 weighted magnetic resonance imaging (MRI) of the supra-cervical FM could predict PROM and PPROM.This prospective cohort study enrolled 77 women between the 28th and 37th weeks of gestation. Two indicators of fetal membrane defects, including prolapsed depth >5 mm and signal abnormalities, are investigated for our prediction. Fisher’s exact test was used to determine whether prolapsed depth >5 mm and/or signal abnormalities were associated with PROM and PPROM. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated for prolapsed depth >5 mm, signal abnormalities, and the combination of prolapsed depth >5 mm and signal abnormalities.Among 12 women with PROM , 9 had membrane prolapse >5 mm and 5 had FM signal abnormalities. Among 65 women with rupture of membranes at term, 2 had membrane prolapse >5 mm and 1 had signal abnormalities. By Fisher’s exact test both indicators, membrane prolapse >5 mm and signal abnormalities, were associated with PROM  and PPROM . Additionally, membrane prolapse >5 mm, signal abnormalities, and the combination of the two indicators all demonstrated high specificity for predicting PROM  and PPROM .in vivo and may be able to identify women at high risk of PPROM.MRI can distinguish the supra-cervical fetal membrane  During pregnancy, the developing fetus is surrounded by the amnion and chorion, collectively called the fetal membrane (FM). In a healthy pregnancy, the FM remains intact until delivery at term, when the rupture of membranes (ROM) occurs either naturally during labor or is induced by the physician. However, in 10% of term deliveries the FM ruptures before labor onset, which is defined as a prelabor rupture of membranes (PROM) . Participants were included if they were age 18 years or older and had a healthy singleton pregnancy. Participants were excluded if they had a twin pregnancy, contraindications to MRI, BMI over 40, major fetal anomalies, placenta implantation, or cervical cerclage. Between April 2017 and Feb 2020, 82 pregnant women were enrolled in the original study in the Department of Obstetrics and Gynecology Outpatient Clinic at Washington University School of Medicine. Written informed consent was obtained from each participant, including consent to future research. For the prospective cohort study, MRI images of the pelvis were collected at multiple time points during pregnancy. For this study, we used the last examination for each patient, performed between the 28MR images were acquired with a 3.0 Tesla unit . Patients were imaged in the left lateral position with their feet entering the magnet bore first. A 30-channel phased-array torso coil covered the entire pelvis. Each subject underwent MRI sequences that included axial and oblique sagittal planes T2 weighted (T2W) half-Fourier acquisition single-shot turbo spin-echo sequence images. The scanning parameters were as follows: repetition time 1800 ms, echo time 94 ms, matrix 320×650, layer thickness 4.0 mm, slice spacing 0.8 mm, number of layers 30, flip angle 140°. The axial and oblique sagittal T2W scanning fields of view were 400×400 mm and 350×350 mm, respectively. The total scanning time for T2WI was 3 minutes.RadiAnt DICOM viewer  was used to assess MR images of the cervix and FM. The images were analyzed without knowledge of pregnancy outcomes by two radiologists (W. Q. and W. W.) with 10-year and 1-year of experience in abdominal MRI, respectively. In order to examine the observer variability, the images were independently and blindly analyzed by the two observers at two different times with a minimum 60-day interval. The consensus was reached through in-person discussion and review in cases of disagreement. The following parameters of the FM overlying the cervix were evaluated: a prolapsed depth >5 mm, defined as a protrusion of amniotic membranes into the internal cervical os by greater than 5 mm from the shoulder of the original internal os as measured along the lateral border of the funnel Figure 1Clinical data recorded included the following: age, parity, last menstrual period dates, medical disorders, and past obstetric outcomes. Current pregnancy information collected included whether or not cerclage was placed, whether or not the woman was treated with progesterone, and any antepartum complications. Hospital records were reviewed after delivery to determine pregnancy outcome. The principal outcome was PROM, which included any rupture before labor . PPROM i® v. 19.0 for Windows®  and GraphPad Prism . Intra- and inter-observer agreements were calculated by means of the kappa index (κ). Reliability is rated as ‘moderate’ for values between 0.41–0.60, as ‘substantial’ for values between 0.61–0.8 and as ‘excellent’ for values above 0.80 .The pregnancy outcomes were as follow: 12 women had PROM , and 65 women had term ROM  and PPROM . The abilities of these measures to predict PROM and PPROM are presented in This study provides evidence that two FM indicators derived from MRI can be used to predict PROM and PPROM. This is the first study to demonstrate the feasibility of using MRI to assess supra-cervical FM. These two MRI features, membrane prolapse >5 mm and FM signal abnormalities, reflect the membrane stretch under unbalanced load and/or pre-weakened FM, and likely indicate the increased risk of PROM and PPROM.in vivo before delivery to the FM surface area ex vivo after delivery revealed that the FM is stretched in vivo by approximately three-fold and is under approximately 15 mmHg pressure . Nonetheless, our study identified two potential MRI-based risk factors of FM rupture: pre-weakened FM, as reflected by signal abnormalities, and abnormal stretch force upon FM, as reflected by FM prolapse. This study suggests that clinical MRI can be used to identify women at increased risk of PPROM. Further studies with larger sample sizes are needed to evaluate the reproducibility of our findings and provide information for future comparative effectiveness and cost-effectiveness studies. Moreover, further MRI analyses may be able to identify women at high risk of PPROM, so as to provide appropriate antepartum surveillance and antenatal treatment.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The prospective cohort study and this study were approved by the Washington University in St. Louis School of Medicine Institutional Review Board . The patients/participants provided their written informed consent to participate in this study.WQ and PZ designed the experiment. WQ and WeiW evaluated magnetic resonance images. ZS, WenW, PW, and QW collected the data and aided in preparation of the manuscript. RM co-supervised the research. YW obtained funding for the project, supervised the work, and participated in preparation of the manuscript. All authors contributed to the article and approved the submitted version.We thank Deborah Frank for editing the manuscript. We also thank Jessica Chubiz and Megan Steiner for coordinating the clinical team to enroll patients.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Small and Medium Enterprises (SMEs), as productive economic businesses, have a vital role in the economy. The role of SMEs in various countries, especially in third world countries, including Indonesia, is undoubtedly one of the supporting sectors of the economy  of the total business actors in Indonesia; on the other hand, large businesses only have a share of 0–0.01% or about 5,400 units. In terms of employment, micro-businesses absorb around 107.2 million workers (89.2%), small businesses 5.7 million (4.74%), and medium enterprises 3.73 million (3.11%) . However, this increase occurred in the medium business classification at 7.51% (yoy) compared to the previous quarter at 5.5% (yoy). Meanwhile, micro and small enterprises each slowed down by 12.7% (yoy) and 10.4% (yoy) compared to the previous quarter's growth of 13.6% (yoy) and 10.8% (yoy), respectively. Based on business classifications, most of the SME loans were channeled to medium-sized business loans, namely 44.5% and the rest for small businesses at 30.1% and micro enterprises at 25.4%.Based on the results of research conducted by Bank Indonesia, it was identified that one of the constraints of banking in extending credit to SMEs is the limited banking information regarding SMEs that are potential and their eligibility, as well as low levels of financial literacy in SMEs, which have an impact on credit absorption by the banking sector. To improve this performance, it is necessary to increase the knowledge, financial literacy, and decision-making abilities of SME entrepreneurs. Hence, this study aims to develop an empirical model of strategies for improving the organizational performance of SMEs as part of improving company performance to build economic resilience in Indonesia.The development of SMEs cannot be separated from their financial management problems. In running a business, of course, good financial management is needed through good knowledge and skills, which, in this case, there are not many SME players who can make it happen. The majority of SMEs are run by families with a lack of financial stability and difficulties in resolving the problems at hand. SMEs tend to lack the resources to exploit technology, resulting in low efficiency, cannot follow existing best practices, and cannot carry out further analysis to measure their performance. The need to increase knowledge for SMEs is a must to improve performance, financial performance, and overall organizational performance, create innovations, and solve problems faced.Every business organization needs to have knowledge management capabilities to improve its learning competencies (Liao et al., One of the knowledge capabilities that need to be improved and the most important thing related to improving SMEs' financial performance is increasing financial literacy. One of the obstacles for banks in extending credit to SMEs is the limited information from the banking sector regarding these SMEs' potential and feasibility. Increasing financial literacy will impact credit absorption by banks, considering that not all SMEs can take advantage of financial products and services. Strategies to increase financial literacy will also help SMEs in business management, starting from budgeting, planning for saving business funds, and overall financial performance. Furthermore, organizational learning ability through problem-solving activities is one of the most important strategic dynamic abilities (Purwanto et al., For this reason, success to improve the performance of SMEs requires a series of several interrelated variables and influences, starting from the knowledge management capabilities possessed, the level of literacy, which is to improve performance, is mediated by abilities both in terms of creativity, and speed in solving any problems that are encountered. This relationship can be illustrated in This study finds a relationship and a new working model that to improve the organizational performance of SMEs, good financial literacy and knowledge capabilities are required to be mediated by creativity and speed in problem-solving. Improving the performance of SMEs from the financial aspect is not always resolved by assisting in the form of loans, but it requires the ability of SMEs to manage and use them (Prahiawan et al., The model confirms the impact of knowledge management capabilities on organizational performance, consisting of financial and non-financial performance. It is essential to emphasize that both knowledge and innovation are fundamental things that are useful for solving problems, and, if implemented effectively, efficiently, and sustainably, can be used as a competitive advantage. On the other hand, in case we face new problems, and beyond our knowledge, the tools we can rely on are our cognitive abilities, especially our creativity.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objectives: The central sterile supply department (CSSD) provides sterile perineal care sets (SPC sets) for use with patients. However, the SPC sets exceed the standard, and the sterilization process incurs high cost. Therefore, a CSSD nursing team set out to find ways to save costs by providing clean perineal care sets (CPC sets) instead of sterile sets. We examined the rate of catheter-associated urinary tract infection (CAUTI) after using CPC sets, measured the satisfaction of the nursing staff who use the CPC sets, evaluated the decrease in the cost to the hospital. Methods: The CSSD nursing team presented some evidence of the benefits of using the CPC sets to the infection control subcommittee and asked for their approval to use CPC sets instead of SPC sets. After approval by the subcommittee, the CSSD nursing staff began to use CPC sets for patients. The incidence of CAUTI was monitored, and a satisfaction survey of the nurses who used the CPC sets was performed. We compared the costs between the SPC set and the CPC set to determine the cost savings. Results: The CAUTI rate did not change after using CPC sets. The nurses who used the CPC sets indicated no difference in satisfaction between the SPC and CPC sets, and the cost of the CPC set was cheaper than the SPC set (27 Baht per set). Conclusions: In a quality improvement effort, using the CPC set was safe for patients. The users were satisfied with the CPC set and trusted the safety of the instruments. Moreover, using the less expensive CPC sets saved the hospital >700,000 Baht per year.


Each spring and fall billions of songbirds depart on nocturnal migrations across the globe. Theory suggests that songbirds should depart on migration shortly after sunset to maximize their potential for nightly flight duration or to time departure with the emergence of celestial cues needed for orientation and navigation. Although captive studies have found that songbirds depart during a narrow window of time after sunset, observational studies have found that wild birds depart later and more asynchronously relative to sunset than predicted.We used coded radio tags and automated radio-telemetry to estimate the time that nearly 400 individuals from nine songbird species departed their breeding or wintering grounds across North America. We also assessed whether each species was most likely beginning long-distance migratory flights at departure or instead first making non-migratory regional flights. We then explored variation in nocturnal departure time by post-departure movement type, species, age, sex, and season.We found that 90% of individuals from species that were likely initiating long-distance migratory flights departed within 69 min of civil dusk, regardless of species, season, age, or sex. By contrast, species that likely first made non-migratory regional movements away from the migratory destination departed later and more asynchronously throughout the night. Regardless of post-departure movement type, 98% of individuals departed after civil dusk but otherwise showed no preference in relation to twilight phase.Although the presence of celestial orientation cues at civil dusk may set a starting point for departure each night, the fact that species likely beginning long-distance migration departed earlier and more synchronously relative to civil dusk than those first making non-migratory regional movements is consistent with the hypothesis that departing promptly after civil dusk functions to maximize the potential for nightly flight duration and distance. By studying the onset of migration, our study provides baseline information about departure decisions that may enhance our understanding of departure timing throughout migration.The online version contains supplementary material available at 10.1186/s40462-023-00382-5. To synchronize their physiology and behavior with the annual and diel rhythms of the environment, organisms have evolved endogenous time-keeping mechanisms such as circannual and circadian clocks , 2. OrgaMost songbirds migrate nocturnally , and by Despite long-held predictions that songbirds should initiate migratory flights during a narrow window of time after sunset and their demonstrated ability to do so in captivity, observations of individual birds in the wild have repeatedly failed to match theory or captive studies. The general pattern observed in wild songbirds thus far is that although many individuals depart within the first four hours after sunset, departure can occur at any time between sunset and sunrise , 25–34. One clue may be that nearly all field studies of nocturnal departure time have been conducted at stopover sites, rather than at breeding or wintering sites, when birds are first initiating migration for the season (but see ). StopovWe used automated radio telemetry  to estimSetophaga coronata), which may migrate medium distances to the mid-Atlantic or long distances to Central America or the Caribbean (Table Setophaga kirtlandii), we collected data from the same population during both spring and fall migration, and for another, Swainson’s Thrush (Catharus ustulatus), we collected data from the same season, but at two different locations . All individuals were captured in mist nests, aged and sexed when possible [To enhance our ability to draw broad conclusions, we collected data from nine species of migratory songbirds from two wintering sites and four breeding sites in North America Table . All spean Table . For onepossible , and banpossible . Radio tSetophaga ruticilla), Ovenbirds (Seiurus aurocapilla), Swainson’s Warblers (Limnothlypis swainsonii), or Quebec-breeding Swainson’s Thrushes make extensive non-migratory regional movements after departure [Setophaga striata), and Yellow-rumped Warblers breeding at the same Nova Scotia breeding sites as the present study have all been previously documented making non-migratory regional movements for days or weeks after departure [Catharus bicknelli) or Gambel’s White-crowned Sparrows (Zonotrichia leucophrys gambelii). To account for this apparent difference in post-departure movement type between species within our sample, we tracked individuals after departure using the Motus Wildlife Tracking System [Previous light-level geolocator and/or radio-tracking data from the same breeding and wintering sites as the present study provides no clear evidence that Kirtland’s Warblers, American Redstarts . Variation in the density and location of stations outside of the wintering and breeding areas for each species prevented us from definitively determining the distance travelled for each individual on the night of departure. To assess whether departing individuals were most likely initiating non-migratory regional flights or long-distance migratory flights, we estimated net displacement distance for individuals that were detected at least once after departure. We defined net displacement as the total distance moved in the general direction of the eventual migratory destination  each time an individual was detected by a station. To calculate net displacement, we first determined the bearing and great circle distance from the site of departure to the station each individual was detected at using packages “swfscMisc”, “geosphn = 20) or between sunrise and sunset (n = 18), assuming that departure during morning twilight or after sunrise was indicative of birds making short-distance local movements through normal daily activities and/or small home range shifts out of range of the stations but without leaving the breeding or wintering site [n = 3) rather than after astronomical dawn, because the latter is never experienced during the departure period due to Alaska’s extreme northern latitude.At the Alaska breeding site, we used two automated telemetry receivers that were not part of Motus to record departure of Gambel’s White-crowned Sparrows. Time of departure was estimated as the time of last detection at the breeding site . At all ing site . For the−6º), sunset position is easily observable, skylight polarization patterns are at maximum visibility, and a few of the brightest stars become visible. During nautical twilight (−6–−12º), the position of the sun is not visible, skylight polarization gradually dims from 50 to 0% brightness, and dozens of additional stars become visible. During astronomical twilight (−12–−18º), light from the sun is no longer visible and stars eventually reach their maximum brightness [To calculate nocturnal departure time in relation to the time of local sunset, civil dusk, and sun elevation angle, we used the package “suncalc”  in Progrightness , 59, 60.For comparison of nocturnal departure time between species, we qualitatively described obvious differences because the shape of departure time distributions varied widely, violating assumptions of parametric and non-parametric central tendency tests. To test for differences in the variability of nocturnal departure, we used Fligner-Killeen tests. To compare nocturnal departure between seasons in Kirtland’s Warblers and between locations in Swainson’s Thrush, we used Mann-Whitney-Wilcoxon tests. To explore age, sex, and year effects on the timing of nocturnal departure, we used ANOVA or Kruskal-Wallis tests. To estimate cloud cover during the hour surrounding civil dusk for each individual, we downloaded hourly weather data (31 km resolution) for each breeding or wintering site from the Copernicus Climate Change Service’s ERA5 reanalysis . We thenAll individuals included in the analyses initiated departure flights nocturnally and permanently departed their breeding or wintering sites for the season. Tracking data indicated that wintering Kirtland’s Warblers and Ovenbirds typically travelled > 500 km towards the breeding grounds in the first few nights after departure. American Redstarts and Swainson’s Warblers were less frequently detected within the first few nights after departure, making it more difficult to assess progress towards the migratory destination immediately after departure. However, because they generally appeared to move at similar rates to Kirtland’s Warblers and Ovenbirds, and previous tracking data from the same study sites has not provided evidence of regional movements post-departure, we classified them as migrating at departure Fig. A. OveralX2 = 130.7, df = 1, n = 324, P < 0.001). Similarly, species likely beginning migration departed at higher sun elevation angles and more synchronously with respect to sun angle (-13.8 ± 6.7º) than those first making regional movements , but this difference reversed and was no longer significant when using time after civil dusk . As a result, we use time after civil dusk as a measure of departure time hereafter.Although individuals from species likely beginning migration primarily departed during a narrow window of time, this window of time spanned multiple periods of twilight both within and between species. Thus, regardless of post-departure movement type, we found no clear preference for departing during a single period of twilight Fig.  bottom. n = 112, P < 0.001). When comparing Swainson’s Thrush from two different breeding locations, we found that individuals breeding in Quebec (62.4 ± 208.7 min) departed earlier after civil dusk than individuals breeding in Nova Scotia Swainson’s Thrush (117.7 ± 198.8 min), but the difference was not significant .Within each species, we found almost no variation in nocturnal departure time by age, sex, or year, irrespective of the metric used Tables  and 3. Fn = 251, P = 0.087). Among species likely beginning migration at departure, we found no significant difference in departure time between overcast (33.5 ± 33.6 min after civil dusk) and mostly clear nights . Similarly, among species likely first making regional flights, we found no difference in departure times related to cloud cover .To determine if the visibility of celestial cues correlated with nocturnal departure time, we compared departure time on overcast (≥ 95% cloud cover) and mostly clear nights (≤ 25% cloud cover), but we found no evidence that cloud cover affected nocturnal departure time. Across all species, 103 of 396 individuals (26%) departed on overcast nights and 148 (37%) departed on mostly clear nights, but we found no significant difference in departure time on overcast nights (41.4 ± 98.3 min after civil dusk) and mostly clear nights  and Great Reed Warbler  flights that lasted more than four hours began shortly after sunset, while flights lasting less than one hour began variably either before sunset or throughout the period between sunset and sunrise. Thus, although there was some variation in the timing of departure across the entire migration period, the vast majority of long-distance migratory flights by Eurasian Hoopoes and Great Reed Warblers began within a narrow range of time after sunset, which suggests that individuals may depart on migratory flights at relatively consistent times throughout migration. However, even if departure times are relatively consistent throughout migration, it is entirely possible that individuals pursuing either time- or energy-minimizing migration strategies depart shortly after civil dusk and instead differ in the frequency of long-distance and short-distance flights, average flight speed, time of flight cessation, and/or time spent at stopover. Only further study of departure times across entire migrations will be able to determine whether early nocturnal departure is part of a time- or energy-minimizing migration strategy, or whether individuals depart shortly after sunset prior to any expected long-distance flight, regardless of their overall migration strategy.Holding other important factors constant , individuals departing shortly after civil dusk consistently throughout their migrations could fly longer and farther each night and arrive to their migratory destination days earlier than individuals consistently departing several hours later in the middle of the night. If this were true, early nocturnal departure could be one aspect of a time-minimizing migration strategy that songbirds use to ensure timely arrival to their migratory destinations. However, it is generally not known whether songbirds depart at consistent times during migration. Using some of the only data available to address this question, Liechti et al.  found thIn contrast to birds that immediately begin migration after departure, birds that first make regional flights should be under no pressure to maximize nightly flight duration. By leaving later in the night, Swainson’s Thrush, Blackpoll Warblers, and Yellow-rumpled Warblers breeding in Nova Scotia may be able to reap the energetic and survival benefits of nocturnal flight , 19 but To accurately orient and navigate, migratory birds use multiple compasses  that are calibrated with information derived from celestial cues –24, and Despite the lack of a clear preference for departing during a single phase of twilight, the fact that 98% of individuals in our study waited until after civil dusk to depart, even with extreme differences in the timing of civil dusk relative to sunset , indicates that there is something special about civil dusk. At least two possible explanations exist, and the evidence is equivocal. It could be that the level of darkness present at civil dusk, in addition to the circadian clock, is necessary for full expression of migratory behavior, as has been shown in captive Gambel’s White-crowned Sparrows . ExperimOur study reveals the potential power of studying departure from breeding and wintering sites at the beginning of migration. By at least partially controlling for immediate differences in migratory route, proximity to ecological barriers, and distance remaining on migration, we were able to document a long-predicted but seldom observed biological pattern. We observed a remarkable degree of synchrony, relative to civil dusk, in the timing of nocturnal departure by species that were likely initiating migratory flights that was consistent across season, age, and sex. By contrast, we found that species likely carrying out non-migratory regional movements departed later and more asynchronously relative to civil dusk. The fact that nearly all individuals waited until after civil dusk to depart highlights the importance of civil dusk, but it remains unclear if the availability of celestial cues at civil dusk sets a starting point for nightly departure  or whethBelow is the link to the electronic supplementary material.Additional file 1. Table S1. Basic descriptive information for populations included in this study. Age classes include hatch year (HY), after hatch-year (AHY), second-year (SY), after second-year (ASY), and unknown (U).


Scalable fabrication of two-dimensional (2D) arrays of quantum dots (QDs) and quantum rods (QRs) with nanoscale precision is required for numerous device applications. However, self-assembly–based fabrication of such arrays using DNA origami typically suffers from low yield due to inefficient QD and QR DNA functionalization. In addition, it is challenging to organize solution-assembled DNA origami arrays on 2D device substrates while maintaining their structural fidelity. Here, we reduced manufacturing time from a few days to a few minutes by preparing high-density DNA-conjugated QDs/QRs from organic solution using a dehydration and rehydration process. We used a surface-assisted large-scale assembly (SALSA) method to construct 2D origami lattices directly on solid substrates to template QD and QR 2D arrays with orientational control, with overall loading yields exceeding 90%. Our fabrication approach enables the scalable, high fidelity manufacturing of 2D addressable QDs and QRs with nanoscale orientational and spacing control for functional 2D photonic devices. Dehydration and surface-assisted assembly enable rapid, scalable quantum dot and quantum rod 2D arrays with nanoscale precision. Quantum dots (QDs) and quantum rods (QRs) exhibit appealing features of bright and tunable narrowband photoluminescence (PL) emission that have attracted extensive interest in the wake of numerous successful state-of-the-art device applications  to promote surface diffusion and self-organization  DNA origami  from their original organic solvent to aqueous buffer, which substantially shortens the time required for their synthesis from a few days to a few minutes. This approach can be applied to QDs and QRs with various sizes, aspect ratios, spectra, and shell surfaces . The dQDface DNA .et al. , commercial QDs/QRs dispersed in an organic solvent are initially mixed with an aqueous solution containing an excess thiolated ssDNA and sodium salt, forming a phase separated liquid double layer . The mix+ and phase transfer catalyst, dehydration volume ratio, and dehydration time on the DNA density per dQD/dQR. Commercial organic 6- and 14-nm CdSe/ZnS QDs with emission wavelengths of 600 (QD600) and 660 nm (QD660), respectively, and 4/16 nm (diameter/length) and 5/29 nm CdSe/CdS QRs with emission wavelengths of 560 (QR560) and 620 nm (QR620), respectively, were selected to prepare dQD600, dQD660, dQR560, and dQR620 using dehydration-assisted DNA conjugation (O-(2-mercaptoethyl)-O′-methyl-hexa(ethylene glycol) (mPEG) and then conjugated with thiolated DNA (mQDs/mQRs) . These results showed that our approach substantially increases DNA surface density up to 10-fold compared with the traditional method , which may be due to the greater ZnS shell thickness of QD660 that may decrease the effect of surface defects and ligands. In addition, we found two key factors to prepare high-density DNA functionalization of QDs and QRs, namely, Na+ and 1-butanol/water volume ratio. An AGE assay was used to characterize DNA loadings on QD660 based on increased gel retardation. We first prepared dQD660 using thiolated DNA (51 nt) with or without NaOH and the phase transfer catalysts trioctylphosphine oxide (TOPO) and tetrabutylammonium bromide (TBAB). We first tested NaOH instead of NaCl as a control, as NaOH has been shown to be necessary in the previous QD/QR phase transfer protocols  . By compDs/mQRs) . Considerotocols . Meanwhirotocols . The incMono-mercapto ligand capped QDs/QRs usually suffer from poor colloidal stability due to dynamic thiol-ZnS or thiol-CdS interactions (n = 3) for QD600-Cy5, QD660-Cy5, QR560-Cy5, and QR620-Cy5, respectively, indicating an increasing number of dye acceptors due to the high-density of DNA functionalization on the QD/QR surface  at the 3′ terminus . For com surface . We esti surface . It shou surface .Having demonstrated the highly efficient hybridization ability of dQDs and dQRs, we next sought to test the loading efficiency of dQDs/dQRs onto DNA origami structures. A rigid 6HB wireframe rhombic DNA origami (Rh) was designed using ATHENA . MoreoveHaving demonstrated excellent colloidal stability and hybridization ability of dQDs/dQRs, we next sought to develop a strategy to fabricate QD/QR 2D arrays. Here, we used Rh to demonstrate a SALSA strategy to fabricate DNA origami lattices directly on a solid substrate with control over relative origami orientation. The key to achieve relative orientational control of origami tiles within a 2D lattices is to introduce anisotropic lateral interactions between neighboring origamis . Althoug+ mediated SALSA where the monovalent cation promotes the diffusion and assembly of origami tiles; (ii) thermal annealing to break misassembled origami tiles for error correction; and (iii) controlling the same side of the origami landing on the substrate. Without or with a low concentration (<100 mM) of Na+, the affinity of the origami to the mica surface was so high that the origami tiles could not diffuse to form ordered lattices once they attached to the surface (fig. S22). Without sufficient thermal annealing, only small 2D arrays were observed due to misassembled origami tiles (fig. S23). Heating at a higher temperature (>60°C) will start to denature DNA origami and lower the surface coverage (fig. S23). Longer annealing times also help to produce large 2D origami lattices to some extent (fig. S24). We found that the conditions described in Methods yielded the best 2D lattices observed thus far.We discovered three synergistic effects that are key to the formation of large origami lattices: (i) Na+ mediating the binding affinity and possibly the elevated temperature promoting dynamics, this method was able to bias the nonbinding face of the origami landing on the mica surface  and up to 73% of QRs were distributed within 30° in a 1-μm2 area using the zipper-like hybridization mode (2 area (fig. S31). This result shows that the zipper-like geometry can form rigid binding by reducing the distance between QRs and DNA nanostructures, which is consistent with results from previous reports  due to uncontrolled nucleation of origami lattices on the surface with random initial orientations and positions. Future work might extend our approach using lithographically defined substrates to manipulate the initial nucleation steps of SALSA. The flexible and accurate angle control of the 6HBs in wireframe origami structures will also enable us to precisely tune the orientation of anisotropic functional materials like nanorods and explore their arrangement-dependent properties. Together, the use of dehydration-assisted DNA conjugation and SALSA is expected to facilitate the translation of nanoscale DNA origami design strategies into surface nanofabrication, where scalable production and nanoscale precision are essential to achieve target device performance.In the current implementation, 2D origami lattices were limited in size , and tris  were purchased from Life Technologies Corporation DBA Invitrogen. Mica discs  were purchased from Ted Pella Inc. and used for all AFM imaging.CdSe/ZnS core/shell QDs [catalog numbers: 900218 (QD600) and 900249 (QD660)], CdSe/CdS core-shell type QRs [catalog numbers: 900512 (QR560) and 900514 (QR620)], MPA , mPEG  were purchased from Sigma-Aldrich. Basic Agarose  was purchased from IBI Scientific. TBE 10× buffer [(pH 8.3) catalog number: 1610733] was purchased from Bio-Rad Laboratories Inc. TAE 10× buffer  and phosphate-buffered saline (PBS) 1× buffer  were purchased from Corning. All DNA oligonucleotides were purchased from Integrated DNA Technologies  with standard desalting or high-performance liquid chromatography (for dye-modified DNA oligonucleotides) as purification method. Thiol-modified oligos ordered from IDT are shipped in their oxidized form, with the sulfur atoms protected by an S─S bond (Mod Code: /5ThioMC6-D/ or /3ThioMC3-D/), and were reduced using TCEP in 100× excess. All DNA oligonucleotides were received as dry pellets. Sodium chloride , MgClg for several seconds to facilitate a liquid phase separation. DNA-functionalized QDs/QRs were then recovered as a sublayer of the resulting two immiscible liquids. To remove excess ssDNA, DNA-functionalized QDs/QRs were purified and concentrated using an ultracentrifugal filter (Amicon 100 kDa) five times at 8000g for 3 min for each centrifugation step. The whole process was carried out under ambient conditions, assisted only by sonication, vortex mixing, and centrifugation-facilitated phase separation.dQDs/dQRs were prepared using dehydration-assisted directly phase transfer from commercial organic QDs/QRs. Briefly, 5 μl of QD  was incubated with thiolated ssDNA (21 nt) (sequence: 5′-thiol-AAA AAA AAA CCC AGG TTC TCT-3′) at a desired molar ratio  in the presence of 100 mM NaOH or NaCl to reach a final volume of 50 μl. Such solution was sonicated at 37 Hz around 5 min and then immediately combined with 600 μl of 1-butanol followed by a quick vortex for several seconds. Subsequently, 200 μl of 0.5× TBE buffer was added to the above solution followed by another quick vortex and a brief centrifugation at 2000g for several seconds, and the aqueous layer was recovered. The vortexing and centrifugation steps were repeated until all aqueous layers were collected. MPA-QDs were purified to remove excess MPA and concentrated using an ultracentrifugal filter (Amicon 30 kDa) six times at 8000g for 5 min for each centrifugation step. After purification, the MPA-QDs were diluted to 500 μl with nuclease-free water and incubated with 20 μl of mPEG for 4 days at room temperature. The MPA/mPEG-QDs were purified and concentrated using an ultracentrifugal filter (Amicon 30 kDa) six times at 8000g for 5 min and then buffer-exchanged into 10 mM tris using a NAP-5 desalting column . Aqueous QDs were incubated with thiolated-ssDNA at desired molar concentration for conjugation . Last, purified DNA-conjugated QDs/QRs concentrations were determined by measuring the absorbance of samples at 350 nm.Aqueous QDs were prepared as described previously, with minor modifications  five times at 8000g for 3 min for each centrifugation step.Aqueous QD660 was prepared as described above. mdQD660 was prepared using dehydration-assisted DNA condense from aqueous QD660. Briefly, 5 μl of QD660 (100 nM) was incubated with thiolated ssDNA (51 nt) (sequence: 5′-thiol-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA TTG TGA CAG CTG GAT CGT TAC-3′) at a desired molar ratio (500:1) in the presence of 100 mM NaOH to reach a final volume of 50 μl. Such solution was sonicated at 37 Hz around 5 min and then immediately combined with 600 μl of 1-butanol followed by a quick vortex for several seconds. Subsequently, 200 μl of 0.5× TBE buffer was added to the above solution followed by another quick vortex and a brief centrifugation at 2000g for several seconds to facilitate a liquid phase separation. DNA-functionalized QD660 were then recovered as a sublayer of the resulting two immiscible liquids. DNA-functionalized QD660 sample without purification (15 μl) was combined with 3 μl of 6× loading buffer (New England Biolabs) and loaded to a 1% agarose gel with 0.5× TBE. Each gel was run at 65 V for 60 min in 0.5×TBE at 4°C. Gels were then visualized under blue light transilluminator.To investigate the impact of NaOH, TOPO, and TBAB on DNA density per QD, dQD660 was incubated with thiolated DNA (51 nt) (sequence: 5′-thiol-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA TTG TGA CAG CTG GAT CGT TAC-3′) (case 1), or combined with NaOH (case 2), or NaOH and TOPO (case 3), or NaOH and TBAB (case 4), or NaOH, TOPO, and TBAB (case 5) before sonication. In a typical experiment, for above case 1, 20 μl of thiolated ssDNA was added to 5 μl of octadecylamine capped QD660 at a molar ratio of 500:1 to reach a final volume of 50 μl. For above case 2, 20 μl of thiolated ssDNA was added to 5 μl of octadecylamine capped QD660 at a molar ratio of 500:1 in the presence of 100 mM NaOH to reach a final volume of 50 μl. For above case 3, 5 μl of octadecylamine capped QD660 was incubated with 5 μl of TOPO (1 g/10 ml) for 30 min, and 20 μl of thiolated ssDNA was added to above mixture at a molar ratio of 500:1 in the presence of 100 mM NaOH to reach a final volume of 50 μl. For above case 4, 5 μl of octadecylamine capped QD660 was incubated with 2 μl of TBAB (0.3 M) for 30 min, and 20 μl of thiolated ssDNA was added to above mixture at a molar ratio of 500:1 in the presence of 100 mM NaOH to reach a final volume of 50 μl. For above case 5, 5 μl of octadecylamine capped QD660 was incubated with 5 μl of TOPO (1 g/10 ml) and 2 μl of TBAB (0.3 M) for 30 min, and 20 μl of thiolated ssDNA was added to above mixture at a molar ratio of 500:1 in the presence of 100 mM NaOH to reach a final volume of 50 μl. Such solution was sonicated around 5 min and then was immediately combined with 600 μl of 1-butanol followed by a quick vortex for several seconds. Subsequently, 200 μl of 0.5× TBE buffer was added to the above solution followed by another quick vortex and a brief centrifugation at 2000g for several seconds to facilitate a liquid phase separation. DNA-functionalized QD660 were then recovered as a sublayer of the resulting two immiscible liquids. DNA-functionalized QD660 sample without purification (15 μl) was combined with 3 μl of 6× loading buffer (New England Biolabs) and loaded to a 1% agarose gel with 0.5× TBE. Each gel was run at 65 V for 60 min in 0.5× TBE at 4°C. Gels were then visualized under blue light transilluminator.To investigate the impact of dehydration volume ratio on DNA density per QD, different 1-butanol/water volume ratio was used to prepare the dQD660 during dehydration process. In a typical experiment, thiolated ssDNA (51 nt) (sequence: 5′-thiol-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA TTG TGA CAG CTG GAT CGT TAC-3′) was added to octadecylamine capped QD660 at a molar ratio of 500:1 in the presence of 100 mM NaOH to reach a final volume of 50 μl. Such solution was sonicated around 5 min and then was immediately combined with 50, 300, 450, 600, or 750 μl of 1-butanol followed by a quick vortex for several seconds. Subsequently, 20, 100, 150, 200, or 250 μl of 0.5×TBE buffer was added to the above solution followed by another quick vortex and a brief centrifugation at 2000g for several seconds to facilitate a liquid phase separation. DNA-functionalized QD660 were then recovered as a sublayer of the resulting two immiscible liquids. DNA-functionalized QD660 sample without purification (15 μl) was combined with 3 μl of 6× loading buffer (New England Biolabs) and loaded to a 1% agarose gel with 0.5× TBE. Each gel was run at 65 V for 60 min in 0.5× TBE at 4°C. Gels were then visualized under blue light transilluminator.Various dehydration time was tested for dQD660 preparation. In a typical experiment, thiolated ssDNA (51 nt) (sequence: 5′-thiol-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA TTG TGA CAG CTG GAT CGT TAC-3′) was added to octadecylamine capped QD660 at a molar ratio of 500:1 in the presence of 100 mM NaOH to reach a final volume of 50 μl. Such solution was sonicated around 5 min and then was immediately combined with 600 μl of 1-butanol followed by a quick vortex for several seconds. After 0, 30, 60, or 90 min of incubation and shaking, 200 μl of 0.5×TBE buffer was added to the above solution followed by another quick vortex and a brief centrifugation at 20006 M−1 cm−1, 2.9 × 107 M−1 cm−1, 2.3 × 107 M−1 cm−1, and 6.4 × 107 M−1 cm−1. DNA concentration was determined by FAM fluorescence calibration curve, where fluorescence was excited at 485 nm with emission recorded from 500 to 700 nm. For comparison, DNA-functionalized QDs/QRs (mQDs/mQRs) were also prepared by a method as described above.To quantify DNA density per QD/QR, 5′-thiolated DNA with an extra FAM at the 3′ terminus (21 nt) (sequence:5′-thiol-AAA AAA AAA CCC AGG TTC TCT-FAM-3′) was used to prepare DNA-conjugated QD/QR. The concentrations of QDs/QRs were obtained by ultraviolet-visible (UV-vis) extinction spectroscopy with diameter-dependent extinction coefficients calculated from an empirical equation (2).To fabricate mQD600-Cy5, dQD600-Cy5, mQD660-Cy5, dQD660-Cy5, mQR560-Cy5, dQR560-Cy5, mQR620-Cy5, and dQR620-Cy5 FRET pairs, mQD600, dQD600, mQD660, dQD660 mQR560, dQR560, mQR620, and dQR620 were incubated with twofold excess complementary DNA with a Cy5 modifier at the 3′ terminus (sequence: 5′-AGA GAA CCT GGG-Cy5–3′) in PBS buffer. After 2-hour incubation, the fluorescence emission spectra of QDs/QRs alone and in the presence of Cy5 were recorded. To fabricate the QD/QR-DNA origami assemblies, wireframe rhombic origami with binding overhangs was incubated with mQD660, dQD660, mQR620, and dQR620 at room temperature overnight, respectively . One hundred microliters of 100 nM DNA scaffold  was mixed with 20 equivalent all corresponding staple strands, and the final buffer condition was adjusted to in 1× TMg. The final concentration of the scaffold was about 20 nM. The mixed solution was annealed in a polymerase chain reaction thermocycler: 95°C for 5 min, 85°C down to 76°C for 5 min/°C, 75°C down to 30°C for 13.75 min /0.5°C, 29°C down to 25°C for 10 min/°C, and then held at room temperature. The crude origami sample was directly used for SALSA without further purification. All DNA sequences are summarized in tables S6 and S7.The 6HB wireframe rhombic origami was folded using a reported method  was placed on top of the liquid surface in the well, floating with the cleaved side in contact with the solution surface. Then, the microplate was sealed and placed on a hotplate shaker  for 12 cycles of heating at 60°, 55°, and 50°C for 1 hour each  with 200 rpm shaking and then let the setup naturally cool down to room temperature. Note that heating temperatures above were instrument settings, and actual sample temperatures were measured to be 50°, 47°, and 44°C, respectively. The mica disc was then taken out of the microplate well and carefully rinsed with 100 μl of 1× TMg buffer (with 0.5 M Na+) 10 times, with 1× TMg buffer (without Na+) 6 times, and with 1× TNi (40 mM tris and 12.5 mM MgCl2 with a pH adjusted to 8.3 ± 0.2) 3 times before incubating with 50 μl 1× TNi on the disc for 5 to 10 min. After the incubation, the disc was rinsed with 100 μl of Milli-Q water three times and dried with compressed air. Next, the mica disc was kept under vacuum for at least 1 hour before AFM imaging. For QD/QR binding experiments, the SALSA sample on the mica disc after annealing was only rinsed 12 times with 100 μl of 1× TMg buffer (with 0.5 M Na+) and kept wet before the next step. Iterations of the synthesis method for optimization were noted with the results in the Supplementary Materials.In a typical SALSA process, the as-synthesized origami was mixed with a concentrated NaCl solution (5 M) for a 1.5-ml solution with a final origami concentration of 500 pM and a Na+) containing 1 nM dQDs/dQRs in a well of a 24-well microplate and incubated for 4 hours with 200 rpm shaking. The mica disc was then taken out of the microplate well and carefully rinsed with 100 μl of 1× TMg buffer (with 0.5 M Na+) 10 times, with 1× TMg buffer (without Na+) 6 times, and with 1× TNi (40 mM tris and 12.5 mM MgCl2 with a pH adjusted to 8.3 ± 0.2) 3 times before incubating with 50 μl of 1× TNi on the disc for 5 to 10 min. After the incubation, the disc was rinsed with 100 μl of Milli-Q water three times and dried with compressed air. dQDs/dQRs were functionalized with a 5′-thiol–modified DNA (sequence: 5′-thiol-AAA AAA AAA CCC AGG TTC TCT-3′) for a “shear-like” hybridization or a 3′-thiolated DNA (sequence: 5′-CCC AGG TTC TCT AAA AAA AAA-thiol-3′) for a “zipper-like” hybridization. For orientation analysis of QR 2D arrays, only the angles of QR monomers in the AFM images were measured and counted in the analysis. OrientationJ (Distribution function (9-μm2 images) or the Measure function (1-μm2 images) with the horizontal x axis as 0°.After rinsing, SALSA origami lattices on a mica disc were placed on the liquid surface of 1 ml of 1× TMg buffer  was drop casted on 400-mesh carbon film square grids . For DNA origami and QD/QR-origami assemblies, 10 μl of wireframe DNA origami objects with or without attached QDs/QRs (5 nM) was adsorbed on glow-discharged 400-mesh carbon film square grids and stained by 2% aqueous uranyl formate solution containing 25 mM NaOH.k = 0.4 N/m, fo = 70 kHz; Bruker) or on an Asylum Research Jupiter XR AFM (Oxford Instruments) in tapping mode using an ARROW-UHF ultrahigh-frequency probe .AFM measurements were performed under air condition in either on an Icon Atomic Force Microscope (Bruker) in ScanAsyst mode using a ScanAsyst-Air silicon tip on nitride lever  were measured using a multimode microplate reader (Tecan Spark). Quantum yields of QDs/QRs were determined using the relative quantum yield determination method with rhodamine 101 in spectroscopic-grade ethanol as standard  (Absorbance spectra were measured using an Evolution 260 Bio UV-vis spectrophotometer (Thermo Fisher Scientific), and steady-state emission spectra (λJ) and Förster distance (R0) were calculated using A(λ) is the molar absorptivity spectrum of the acceptor in M−1 cm−1, and λ is the wavelength in nm (2 is the orientation factor (assumed to be 2/3), ΦD is the quantum yield of the donor, and n = 1.35 is the refractive index of the medium. The molar extinction coefficients for Cy5 were obtained from the suppliers.The overlap integral  is circularly polarized with a quarter-wave plate (AQWP05M-600) to ensure uniform excitation. The emission is filtered with a Semrock BrightLine 409-nm longpass filter to remove the laser light, and then sample emission passes through a half-wave plate (HWP) (AHWP05M-600) and a wire-grid polarizer (WP25M-VIS). For acquiring energy spectra, the light was directed into a Princeton Instruments Acton spectrometer and a Princeton Instruments ProEM 512 × 512 charge-coupled device array. As the HWP is rotated, the change intensity is recorded by integrating the entirety of the spectral range (roughly 533 to 700 nm), as it passes through the parallel (perpendicular) polarizer, and the degree of polarization at each detection angle is calculated using