Perspectives on label-free microscopy of heterogeneous and dynamic biological systems.

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm2 that is well below damage thresholds (25 J/cm2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent versus activated (CD3/CD28/CD2) primary human T cells, and quiescent versus activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

Rotation from methadone to buprenorphine using a micro-dosing regime in patients with opioid use disorder and serious mental illness: A case series.

INTRODUCTION: Inducting buprenorphine from methadone has traditionally involved initial opioid withdrawal, with risk of mental state deterioration in patients with serious mental illness (SMI). Micro-dosing of buprenorphine, with small incremental doses, is a novel off-label approach to transitioning from methadone and does not require a period of methadone abstinence. Given the limited literature about buprenorphine microdosing, we aimed to evaluate the feasibility and safety of inducting buprenorphine in a series of patients on methadone with SMI. METHODS: For this retrospective case series, we reviewed the records of 16 patients with SMI at a Melbourne addiction treatment centre, from January 2021 to July 2022, who transitioned via micro-dosing, from high-dose methadone (>30 mg) to buprenorphine and depot-buprenorphine. Psychiatric diagnoses, mental state, other substance withdrawal, transfer success, transition time, opioid withdrawal symptoms and overall patient experience were collected via objective and subjective reporting. RESULTS: Methadone to buprenorphine transfer was completed by 88% of patients. Mental health measures remained stable with the exception of mildly increased anxiety. Median transfer time was 6.5 days for inpatients, 9 days for mixed setting and 10 days for outpatients. Most patients (93%) rated their experience 'manageable' reporting mild withdrawal symptoms. One patient met study criteria for precipitated withdrawal. DISCUSSION AND CONCLUSIONS: This retrospective case series provides evidence that the use of a micro-dosing buprenorphine induction for methadone to buprenorphine transitions, including to depot-buprenorphine, has negligible risk, is tolerated by patients with SMI and is unlikely to precipitate an exacerbation of their mental illness.

Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence.

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex, with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population, a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro, and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells, MP TRAC-CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity, and reduced IFN-γ, IL-2, IP-10, IL-1ß, IL-17, and TGF-ß production at the end of manufacturing ex vivo, with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo, which could lead to better responses against solid tumors in vivo.

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375nm picosecond-pulsed diode laser operating at 50MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification ( i.e ., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5ms integration time per cell, resulting in a light dose of 2.65 J/cm 2 that is well below damage thresholds (25 J/cm 2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent vs. activated (CD3/CD28/CD2) primary human T cells, and quiescent vs. activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

Disparities in incidence and survival for patients with Ewing sarcoma in Florida.

BACKGROUND: Ewing sarcoma (ES) is a malignant bone tumor most commonly affecting non-Hispanic White (NHW) adolescent males, though recognition among Hispanic individuals is rising. Prior population-based studies in the United States (US), utilizing Surveillance, Epidemiology, and End Results (SEER) have shown higher all-cause mortality among White Hispanics, Blacks, and those of low socioeconomic status (SES). Florida is not part of SEER but is home to unique Hispanic populations including Cubans, Puerto Ricans, South Americans that contrasts with the Mexican Hispanic majority in other US states. This study aimed to assess racial/ethnic disparities on incidence and survival outcomes among this diverse Florida patient population. METHODOLOGY: Our study examined all patients diagnosed with osseous ES (2005-2018) in Florida (n = 411) based on the state's population-based cancer registry dataset. Florida Age-adjusted Incidence Rates (AAIRs) were computed by sex and race-ethnicity and compared to the equivalent populations in SEER. Cause-specific survival disparities among Florida patients were examined using Kaplan-Meier analysis. Univariable and multivariable analyses using Cox regression were performed for race/ethnicity, with adjustment for age, sex, year of diagnosis, site of disease, staging, SES, and insurance type. RESULTS: There was a significantly higher incidence of osseous ES in Florida Hispanic males (AAIR 2.6/1,000,000); (95% CI: 2.0-3.2 per 1,000,000; n = 84) compared to the SEER Hispanic males (AAIR 1.2/1,000,000;1.1-1.4 per 1,000,000; n = 382). Older age, distant metastasis, lack of chemotherapy or surgical resection were statistically significant determinants of poor survival while SES, insurance status and race-ethnicity were not. However, among nonmetastatic ES, Florida Hispanics had an increased risk of death compared to Florida NHW (adjusted Hazard Ratio 2.32; 95%CI: 1.20-4.46; p = 0.012). CONCLUSIONS: Florida Hispanic males have a higher-than-expected incidence of osseous ES compared to the US. Hispanics of both sexes show remarkably worse survival for nonmetastatic disease compared to NHW. This disparity is likely multifactorial and requires further in-depth studies.

Metabolic priming of GD2 TRAC -CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence.

Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact T-cell phenotypes. CRISPR/Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant ( TRAC ) gene resulting in TRAC -CAR T cells with an enriched stem cell memory T-cell population, a process that could be further optimized through modifications to the media composition. In this study we generated anti-GD2 TRAC -CAR T cells using "metabolic priming" (MP), where the cells were activated in glucose/glutamine low media and then expanded in glucose/glutamine high media. T cell products were evaluated using spectral flow cytometry, metabolic assays, cytokine production, cytotoxicity assays in vitro and potency against human GD2+ xenograft neuroblastoma models in vivo . Compared to standard TRAC -CAR T cells, MP TRAC -CAR T cells showed less glycolysis, higher CCR7/CD62L expression, more bound NAD(P)H activity and reduced IFN-γ, IL-2, IP-10, IL-1ß, IL-17, and TGFß production at the end of manufacturing ex vivo , with increased central memory CAR T cells and better persistence observed in vivo . Metabolic priming with media during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo , which could lead to better responses against solid tumors in vivo .

Novel multimodal cation-exchange membrane for the purification of a single-chain variable fragment from Pichia pastoris supernatant.

A novel salt-tolerant cation-exchange membrane, prepared with a multimodal ligand, 2-mercaptopyridine-3-carboxylic acid (MMC-MPCA), was examined for its purification properties in a bind-and-elute mode from the high conductivity supernatant of a Pichia pastoris fermentation producing and secreting a single-chain variable fragment (scFv). If successful, this approach would eliminate the need for a buffer exchange prior to product capture by ion-exchange. Two fed-batch fermentations of Pichia pastoris resulted in fermentation supernatants reaching an scFv titer of 395.0 mg/L and 555.7 mg/L, both with a purity of approximately 83 %. The MMC-MPCA membrane performance was characterized in terms of pH, residence time (RT), scFv load, and scFv concentration to identify the resulting dynamic binding capacity (DBC), yield, and purity achieved under optimal conditions. The MMC-MPCA membrane exhibited the highest DBC of 39.06 mg/mL at pH 5.5, with a residence time of 1 min, while reducing the pH below 5.0 resulted in a significant decrease of the DBC to around 2.5 mg/mL. With almost no diffusional limitations, reducing the RT from 2 to 0.2 min did not negatively impact the DBC of the MMC-MPCA membrane, resulting in a significant improvement in productivity of up to 180 mg/mL/min at 0.2 min RT. Membrane fouling was observed when reusing the membranes at 0.2 and 0.5 min RT, likely due to the enhanced adsorption of impurities on the membrane. Changing the amount of scFv loaded onto the membrane column did not show any changes in yield, instead a 10-20 % loss of scFv was observed, which suggested that some of the produced scFv were fragmented or had aggregated. When performing the purification under the optimized conditions, the resulting purity of the product improved from 83 % to approximately 92-95 %.

In vitro and in vivo anti-tumor effect of Trichobakin fused with urokinase-type plasminogen activator ATF-TBK.

BACKGROUND: Trichobakin (TBK), a member of type I ribosome-inactivating proteins (RIPs), was first successfully cloned from Trichosanthes sp Bac Kan 8-98 in Vietnam. Previous study has shown that TBK acts as a potential protein synthesis inhibitor; however, the inhibition efficiency and specificity of TBK on cancer cells remain to be fully elucidated. METHODS AND RESULTS: In this work, we employed TBK and TBK conjugated with a part of the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), which contains the Ω-loop that primarily interacts with urokinase-type plasminogen activator receptor, and can be a powerful carrier in the drug delivery to cancer cells. Four different human tumor cell lines and BALB/c mice bearing Lewis lung carcinoma cells (LLC) were used to evaluate the role of TBK and ATF-TBK in the inhibition of tumor growth. Here we showed that the obtained ligand fused RIP (ATF-TBK) reduced the growth of four human cancer cell lines in vitro in the uPA receptor level-dependent manner, including the breast adenocarcinoma MDA-MB 231 cells and MCF7 cells, the prostate carcinoma LNCaP cells and the hepatocellular carcinoma HepG2 cells. Furthermore, the conjugate showed anti-tumor activity and prolonged the survival time of tumor-bearing mice. The ATF-TBK also did not cause the death of mice with doses up to 48 mg/kg, and they were not significantly distinct on parameters of hematology and serum biochemistry between the control and experiment groups. CONCLUSIONS: In conclusion, ATF-TBK reduced the growth of four different human tumor cell lines and inhibited lung tumor growth in a mouse model with little side effects. Hence, the ATF-TBK may be a target to consider as an anti-cancer agent for clinical trials.

Tracing cancer evolution and heterogeneity using Hi-C.

Chromosomal rearrangements can initiate and drive cancer progression, yet it has been challenging to evaluate their impact, especially in genetically heterogeneous solid cancers. To address this problem we developed HiDENSEC, a new computational framework for analyzing chromatin conformation capture in heterogeneous samples that can infer somatic copy number alterations, characterize large-scale chromosomal rearrangements, and estimate cancer cell fractions. After validating HiDENSEC with in silico and in vitro controls, we used it to characterize chromosome-scale evolution during melanoma progression in formalin-fixed tumor samples from three patients. The resulting comprehensive annotation of the genomic events includes copy number neutral translocations that disrupt tumor suppressor genes such as NF1, whole chromosome arm exchanges that result in loss of CDKN2A, and whole-arm copy-number neutral loss of homozygosity involving PTEN. These findings show that large-scale chromosomal rearrangements occur throughout cancer evolution and that characterizing these events yields insights into drivers of melanoma progression.

Human visceral adipose tissue microvascular endothelial cell isolation and establishment of co-culture with white adipocytes to analyze cell-cell communication.

Communication between adipocytes and endothelial cells (EC) is suggested to play an important role in the metabolic function of white adipose tissue. In order to generate tools to investigate in detail the physiology and communication of EC and adipocytes, a method for isolation of adipose microvascular EC from visceral adipose tissue (VAT) biopsies of subjects with obesity was developed. Moreover, mature white adipocytes were isolated from the VAT biopsies by a method adapted from a previously published Membrane aggregate adipocytes culture (MAAC) protocol. The identity and functionality of the cultivated and isolated adipose microvascular EC (AMvEC) was validated by imaging their morphology, analyses of mRNA expression, fluorescence activated cell sorting (FACS), immunostaining, low-density lipoprotein (LDL) uptake, and in vitro angiogenesis assays. Finally, we established a new trans filter co-culture system (membrane aggregate adipocyte and endothelial co-culture, MAAECC) for the analysis of communication between the two cell types. EC-adipocyte communication in this system was validated by omics analyses, revealing several altered proteins belonging to pathways such as metabolism, intracellular transport and signal transduction in adipocytes co-cultured with AMvEC. In reverse experiments, induction of several pathways including endothelial development and functions was found in AMvEC co-cultured with adipocytes. In conclusion, we developed a robust method to isolate EC from small quantities of human VAT. Furthermore, the MAAECC system established during the study enables one to study the communication between primary white adipocytes and EC or vice-versa and could also be employed for drug screening.

Water lettuce (Pistia stratiotes L.) increases biogas effluent pollutant removal efficacy and proves a positive substrate for renewable energy production.

Background: Aquatic plants play a crucial role in nature-based wastewater treatment and provide a promising substrate for renewable energy production using anaerobic digestion (AD) technology. This study aimed to examine the contaminant removal from AD effluent by water lettuce (WL) and produce biogas from WL biomass co-digested with pig dung (PD) in a farm-scale biogas digester. Methods: The first experiment used styrofoam boxes containing husbandry AD effluent. WLs were initially arranged in 50%, 25%, 12.5%, and 0% surface coverage. Each treatment was conducted in five replicates under natural conditions. In the second experiment, WL biomass was co-digested with PD into an existing anaerobic digester to examine biogas production on a farm scale. Results: Over 30 days, the treatment efficiency of TSS, BOD5, COD, TKN, and TP in the effluent was 93.75-97.66%, 76.63-82.56%, 76.78-82.89%, 61.75-63.75%, and 89.00-89.57%, respectively. Higher WL coverage increased the pollutant elimination potential. The WL biomass doubled after 12 days for all treatments. In the farm-scale biogas production, the biogas yield varied between 190.6 and 292.9 L kg VSadded-1. The methane content reached over 54%. Conclusions: WL removed AD effluent nutrients effectively through a phytoremediation system and generated significant biomass for renewable energy production in a farm-scale model.

Prevalence and impact of myocardial injury among patients hospitalized with COVID-19.

Background: Myocardial injury is a prevalent complication observed in patients hospitalized with COVID-19 and is strongly associated with severe illness and in-hospital mortality. However, the long-term consequences of myocardial injury on clinical outcomes remain poorly understood. This study aimed to assess the impact of myocardial injury on both acute-phase and long-term prognosis in COVID-19 patients. Methods: A retrospective, observational study was conducted on all patients who received treatment at the Intensive Care Center for COVID-19 patient, University Medical Center Ho Chi Minh City (UCICC), from August 3rd, 2021, to October 28th, 2021. Results: A total of 582 patients were enrolled in the study, of which 55.3% were female. The mean age of participants was 63.3 ± 16.2. Out of these patients, 330 cases (56.8%) showed myocardial injury. Compared to patients without myocardial injury, those with myocardial injury were older and had a higher incidence of chronic diseases including hypertension, ischemic heart disease, atrial fibrillation, heart failure, diabetes mellitus, chronic kidney disease. They also presented with more severe respiratory failure upon admission and showed a more pronounced abnormality in inflammation and kidney function tests. Furthermore, the in-hospital mortality rate was significantly higher in the group with myocardial injury (49.7% vs 14.3%, p < 0.001). After adjusting for age, gender, comorbidities, renal function, and disease severity at admission, myocardial injury emerged as an independent risk factor for in-hospital mortality (OR = 3.758, 95% CI 1.854-7.678, p < 0.001). Among successfully discharged COVID-19 patients, the all-cause mortality rate after a median follow-up of 18.4 months was 7.9%. Patients with myocardial injury had a significantly higher long-term mortality rate compared to those without myocardial injury (14.0% vs. 3.2%, p < 0.001). However, multivariable Cox regression analysis did not find myocardial injury to be a significant predictor of long-term mortality (HR = 2.128, 95% CI 0.792-5.712, p = 0.134). Conclusions: Myocardial injury is a common and serious complication in hospitalized COVID-19 patients, associated with increased in-hospital mortality. However, it does not significantly impact long-term mortality in successfully discharged COVID-19 patients.

Qualitative feasibility study of the mobile app Destroke for clinical stroke monitoring based on the NIH stroke scale.

Background: Stroke is a leading cause of severe disability in the United States, but there is no effective method for patients to accurately detect the signs of stroke at home. We developed a mobile app, Destroke, that allows remote performance of a modified NIH stroke scale (NIHSS) by patients. Aims: To assess the feasibility of a mobile app for stroke monitoring and education by patients with a history of stroke. Materials and methods: We enrolled 25 patients with a history of stroke in a prospective open-label study to evaluate the feasibility of the Destroke app in patients with stroke. Nineteen patients completed all study assessments, with a median time from stroke onset to enrollment of 5.6 years (range 0.1-12 years). We designed a modified NIHSS that assessed 12 out of 16 tasks on the NIHSS. Patients completed this test eight times over a 28-day period. We conducted pre-study surveys that assessed demographic information, stroke and cardiovascular history, baseline NIHSS, and experience using mobile technologies, and mid- and post-study surveys that assessed patient satisfaction on app usage and confidence in stroke detection. Results: Ten men and nine women participated in this study (median age of 64 (33-76)), representing ten US states and Washington D.C. Median baseline NIHSS was 0 (0-4). 15 patients reported using health apps. On a 5-point Likert scale, patients rated the app as 4.2 on being able to understand and use the app and 4.3 on using the app when instructed by their doctor. For eight patients with poor confidence in detecting the signs of a stroke before the study, six showed higher confidence after the study. Conclusions: The use of an at-home stroke monitoring app is feasible by patients with a history of stroke and improves confidence in detecting the signs of stroke.

The Germin-like protein gene OsGER4 is involved in heat stress response in rice root development.

Rice (Oryza sativa L.) is one of the most important dietary carbohydrate sources for half of the world's population. However, it is not well adapted to environmental stress conditions, necessitating to create new and improved varieties to help ensure sufficient rice production in the face of rising populations and shrinking arable land. Recently, the development of the CRISPR/Cas9 gene editing system has allowed researchers to study functional genomics and engineer new rice varieties with great efficiency compared to conventional methods. In this study, we investigate the involvement of OsGER4, a germin-like protein identified by a genome-wide association study that is associated with rice root development under a stress hormone jasmonic acids treatment. Analysis of the OsGER4 promoter region revealed a series of regulatory elements that connect this gene to ABA signaling and water stress response. Under heat stress, osger4 mutant lines produce a significantly lower crown root than wild-type Kitaake rice. The loss of OsGER4 also led to the reduction of lateral root development. Using the GUS promoter line, OsGER4 expression was detected in the epidermis of the crown root primordial, in the stele of the crown root, and subsequently in the primordial of the lateral root. Taken together, these results illustrated the involvement of OsGER4 in root development under heat stress by regulating auxin transport through plasmodesmata, under control by both ABA and auxin signaling.

Touch-free optical technologies to streamline the production of T cell therapies.

Currently approved adoptive T cell therapy relies on autologous (obtained from the same patient) T cells, which often suffer from poor quality that diminishes treatment efficacy. Due to the heterogeneous nature of T cell quality between and within patients, significant efforts are aimed at optimizing cell manipulation and growth conditions for potent T cell products. We believe that touch-free imaging and sensing technologies are critical to monitor single-cell features during T cell manufacturing to ensure consistent and optimally timed methods for cell manipulation and growth. Here, we discuss emerging label-free optical imaging and sensing methods, along with machine learning techniques that could enable in-line feedback to optimize T cell quality at multiple stages during manufacturing. These methods have the potential to streamline current workflow, accelerate the manufacture of safe high-quality T cell therapies, and improve our understanding of the dynamic, heterogeneous processes of T cell manufacturing.

Thermally stable cellulose nanospheres prepared from office waste paper by complete removal of hydrolyzed sulfate groups.

Cellulose nanocrystals are commonly obtained by acid hydrolysis, particularly with H2SO4. However, a small amount of deposited sulfate-groups contributes to the degradation of their thermal stability. This study prepared thermally-stable and sulfate-group-free cellulose nanospheres (CNSs) from office waste paper by H2SO4 hydrolysis followed by solvolytic desulfation. The optimal desulfation conditions (i.e., 5 wt% MeOH, reaction temperature of 90 °C, a reaction time of 20 min, 0.5 mM pyridine) were preliminarily found from a one-factor-at-a-time experiment and validated by the results of a central composite design. The optimal desulfation conditions promoted environmental sustainability with less pyridine and MeOH and comparably shorter reaction time. The desulfated CNSs had a significant thermal stability enhancement from 186 to 340 °C. Comprehensive characterization of the morphology, chemical composition, and thermal behavior of the desulfated CNSs reconfirmed the complete removal of sulfate groups without harmful pyridine residues, demonstrating the potential use of the thermally stable CNSs.

The Germin-like protein OsGER4 is involved in promoting crown root development under exogenous jasmonic acid treatment in rice.

In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.

A low-cost, flexible extruder for liposomes synthesis and application for Murrayafoline A delivery for cancer treatment.

Liposomal encapsulation is a drug delivery strategy with many advantages, such as improved bioavailability, ability to carry large drug loads, as well as controllability and specificity towards various targeted diseased tissues. Currently, most preparation techniques require an additional extrusion or filtering step to obtain monodisperse liposomes with the size of less than 100 nm. In this study, a compact liposome extruder was designed at a cost of $4.00 and used to synthesize liposome suspensions with defined particle size and high homogeneity for Murrayafoline A (Mu-A) loading and release. The synthesized MuA-loaded liposomes displayed a biphasic drug release and remained stable under the storage condition of 4°C. They also significantly reduced the viability of HepG2 cells in the cancer spheroids by 25%. The low-cost, flexible liposome extruder would allow the researchers to study liposomes and their applications in a cost-effective manner.

Synthesis of cellulose-g-poly(acrylic acid) with high water absorbency using pineapple-leaf extracted cellulose fibers.

This study synthesized cellulose-g-poly(acrylic acid) with high water absorbency using the cellulose extracted from pineapple leaves. The synthesis experiment used a novel combination of ethylene glycol dimethacrylate (EGDMA) and azobisisobutyronitrile (AIBN) as the cross-linker and the initiator, respectively. Experimental results showed that the concentrations of AIBN and EGDMA had significant effects on the structure and the water absorbency of the cross-linked materials. The cellulose-g-poly(acrylic acid) synthesized with 0.5 wt% AIBN and 0.5 wt% EGDMA had an excellent swelling capacity of 1900 g/g in distilled water, significantly larger than previously reported ones. Compared with poly(acrylic acid), the cross-linked product demonstrated an absorbency improvement of 1.65 times in distilled water and 1.27 times in 8.6 ppm NaCl solution that was the highest salinity level in Ben Tre, Vietnam, in March 2020. Therefore, the obtained product showed high potential for agricultural applications, especially in coastal regions facing a growing thread of saltwater intrusion.