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Mesenchymal stem cells (MSCs) are a type of cell capable of regulating the immune system, as well as exhibiting self-renewal and multi-lineage differentiation potential. Mesenchymal stem cells have emerged as an essential source of seed cells for therapeutic cell therapy. It is crucial to cryopreserve MSCs in liquid nitrogen prior to clinical application while preserving their functionality. Furthermore, efficient cryopreservation greatly enhances MSCs' potential in a range of biological domains. Nevertheless, there are several limits on the MSC cryopreservation methods now in use, necessitating thorough biosafety assessments before utilizing cryopreserved MSCs. Therefore, in order to improve the effectiveness of cryopreserved MSCs in clinical stem cell treatment procedures, new technological techniques must be developed immediately. The study offers an exhaustive analysis of the state-of-the-art MSC cryopreservation techniques, their effects on MSCs, and the difficulties encountered when using cryopreserved MSCs in clinical applications. [ABSTRACT FROM AUTHOR]

Biobanks have become an integral part of health and bioscience research. However, the ultra-low temperature (ULT) storage methods that biobanks employ [ULT freezers and liquid nitrogen (LN2)] are associated with carbon emissions that contribute to anthropogenic climate change. This paper aims to provide a 'Roadmap' for reducing carbon emissions associated with ULT storage in biobanking. The Roadmap offers recommendations associated with nine areas of ULT storage practice: four relating to ULT freezers, three associated with LN2 storage, and two generalised discussions regarding biosample management and centralisation. For each practice, we describe (a) the best approaches to mitigate carbon emissions, (b) explore barriers associated with hindering their implementation, and (c) make a series of recommendations that can help biobank stakeholders overcome these barriers. The recommendations were the output of a one year, UK-based, multidisciplinary research project that involved a quantitative Carbon Footprinting Assessment of the emissions associated with 1 year of ULT storage (for both freezers and LN2) at four different case study sites; as well as two follow up stakeholder workshops to qualitatively explore UK biobank stakeholder perceptions, views, and experiences on how to consider such assessments within the broader social, political, financial, technical, and cultural contexts of biobanking. [ABSTRACT FROM AUTHOR]

Background: Plasmodium vivax is the second most prevalent cause of malaria yet remains challenging to study due to the lack of a continuous in vitro culture system, highlighting the need to establish a biobank of clinical isolates with multiple freezes per sample for use in functional assays. Different methods for cryopreserving parasite isolates were compared and subsequently the most promising one was validated. Enrichment of early- and late-stage parasites and parasite maturation were quantified to facilitate assay planning. Methods: In order to compare cryopreservation protocols, nine clinical P. vivax isolates were frozen with four glycerolyte-based mixtures. Parasite recovery post thaw, post KCl-Percoll enrichment and in short-term in vitro culture was measured via slide microscopy. Enrichment of late-stage parasites by magnetic activated cell sorting (MACS) was measured. Short and long-term storage of parasites at either − 80 °C or liquid nitrogen were also compared. Results: Of the four cryopreservation mixtures, one mixture (glycerolyte:serum:RBC at a 2.5:1.5:1 ratio) resulted in improved parasite recovery and statistically significant (P < 0.05) enhancement in parasite survival in short-term in vitro culture. A parasite biobank was subsequently generated using this protocol resulting in a collection of 106 clinical isolates, each with 8 vials. The quality of the biobank was validated by measuring several factors from 47 thaws: the average reduction in parasitaemia post-thaw (25.3%); the average fold enrichment post KCl-Percoll (6.65-fold); and the average percent recovery of parasites (22.0%, measured from 30 isolates). During short-term in vitro culture, robust maturation of ring stage parasites to later stages (> 20% trophozoites, schizonts and gametocytes) was observed in 60.0% of isolates by 48 h. Enrichment of mature parasite stages via MACS showed good reproducibility, with an average of 30.0% post-MACS parasitaemia and an average of 5.30 × 105 parasites/vial. Finally, the effect of storage temperature was tested, and no large impacts from short-term (7 days) or long-term (7–10 years) storage at − 80 °C on parasite recovery, enrichment or viability was observed. Conclusions: Here, an optimized freezing method for P. vivax clinical isolates is demonstrated as a template for the generation and validation of a parasite biobank for use in functional assays. [ABSTRACT FROM AUTHOR]

Background: Colonial and multicellular volvocine green algae have been extensively studied recently in various fields of the biological sciences. However, only one species (Pandorina morum) has been cryopreserved in public culture collections. Results: Here, we investigated conditions for cryopreservation of the multicellular volvocine alga Gonium pectorale using vegetative colonies or cells and zygotes. Rates of vegetative cell survival in a G. pectorale strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3% and 6%) of the cryoprotectant N,N-dimethylformamide (DMF) and two types of tubes (0.2-mL polymerase chain reaction tubes and 2-mL cryotubes) used for cryopreservation. Among the four conditions investigated, the highest rate of survival [2.7 ± 3.6% (0.54–10%) by the most probable number (MPN) method] was obtained when 2.0-mL cryotubes containing 1.0 mL of culture samples with 6% DMF were subjected to cryogenic treatment. Using these optimized cryopreservation conditions, survival rates after freezing in liquid nitrogen were examined for twelve other strains of G. pectorale and twelve strains of five other Gonium species. We obtained ≥ 0.1% MPN survival in nine of the twelve G. pectorale strains tested. However, < 0.1% MPN survival was detected in eleven of twelve strains of five other Gonium species. In total, ten cryopreserved strains of G. pectorale were newly established in the Microbial Culture Collection at the National Institute for Environmental Studies. Although the cryopreservation of zygotes of volvocine algae has not been previously reported, high rates (approximately 60%) of G. pectorale zygote germination were observed after thawing zygotes that had been cryopreserved with 5% or 10% methanol as the cryoprotectant during two-step cooling and freezing in liquid nitrogen. Conclusions: The present study demonstrated that cryopreservation of G. pectorale is possible with 6% DMF as a cryoprotectant and 1.0-mL culture samples in 2.0-mL cryotubes subjected to two-step cooling in a programmable freezer. [ABSTRACT FROM AUTHOR]

Background: Captive breeding of bonobos (Pan paniscus) has proven to be successful, but maintaining genetic diversity remains a challenge. Cryopreservation of semen is an important potential tool to maintain genetic diversity by preserving current genetic material for future use, as well as facilitating the transport and exchange of genetic material. This study aimed to develop a protocol for semen collection and cryopreservation in the bonobo. Semen was collected from four healthy adult bonobos under general anesthesia during management translocation procedures. Semen collection utilizing urethral catheterization was not successful (n = 1), however, all males (n = 4) responded well to rectal probe electro-ejaculation. Immediately after collection, ejaculates were evaluated for color and admixtures, volume, motility, and concentration. Eosin-Nigrosin staining was prepared to evaluate morphology and viability. Ejaculates were split into two equal volumes and cryopreserved in two different extenders, using a one-step and a two-step approach. Ejaculates were gradually cooled to 4 °C in two hours, subsequently stored in liquid nitrogen vapor for twenty minutes (0.25 ml straws), and finally dropped into liquid nitrogen. Results: Pre-freeze evaluation showed thick, white samples with an average ejaculate volume of 450 µl (100-1000 µl), total motility of 59% (40–80%), viability of 69% (38–85%) and 58% (46–72%) normal spermatozoa. Mainly head (22%) and tail (19%) defects were detected on the Eosin-Nigrosin stain. Ejaculates were highly concentrated, nevertheless, due to the coagulum that caused high viscosity and non-homogenous fractions, only estimations of concentration could be made (1000 million/ml). After 24 h of storage, the post-thaw evaluation showed a loss of quality with an average post-thaw total motility of 15% (5–25%) using the one-step freezing medium, and 19% (5–30%) using the two-step medium. Average post-thaw viability was 15% (4–24%) and 21% (15–29%), respectively. Conclusions: This report on ejaculates from bonobos obtained by rectal probe electro-ejaculation shows that semen parameters of this species are not completely similar to those of its sibling species, the chimpanzee. Further studies are necessary to develop an optimal protocol for the processing and cryopreservation of bonobo spermatozoa. [ABSTRACT FROM AUTHOR]

Background: Long-term preservation of adipose tissue is crucial for clinical applications. Researchers should consider both efficiency and biosafety when choosing a cryoprotective agent (CPA) for adipose tissue preservation. Glycerol has been applied as a nontoxic CPA for multiple tissues but not adipose tissue. We aimed to evaluate the efficacy of glycerol as a CPA for adipose tissue cryopreservation. Methods: Fresh human adipose tissues were obtained from patients who underwent liposuction and divided into 1 mL samples. Each sample was randomly mixed with 1 mL of CPA: 60–100% glycerol, 0.25 mol/L trehalose or DMSO + FBS and cryopreserved in − 196 °C liquid nitrogen for one month. After thawing and elution, the tissues were immediately evaluated for activity and structural integrity in vitro. Then, 0.2 mL of each sample was transplanted subdermally to the nude mouse dorsum and harvested after one month for histological examination to assess the effect of the cryopreserved fat in transplantation. Results: After cryopreservation, the samples treated with DMSO + FBS, trehalose, 60% and 70% glycerol had a more integrated structure than the samples in other groups. Tissues preserved with 70% glycerol had the highest G3PDH activity of 24.41 ± 0.70, comparable to 24.76 ± 0.48 in fresh tissue (p > 0.05). Adipose-derived stem cells (ASC) viability, proliferation and differentiation capability were also better preserved in 70% glycerol group. In vivo analysis showed that tissue preserved with 70% glycerol had a retention rate of 52.37 ± 7.53%, significantly higher than other groups. Histological observation demonstrated better structural integrity and viability in 70% glycerol group. Compared to the DMSO + FBS and trehalose groups, the glycerol groups showed lower tissue inflammation. Conclusion: Glycerol (70%) is efficient in adipose tissue cryopreservation. Glycerol-based CPAs, which are nontoxic and show biosafety, are a promising solution for clinical tissue cryopreservation. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle biopsies are valuable in clinical and research settings, contributing to advancements in diagnosing, understanding, and treating muscle-related conditions. Traditional freezing methods often cause artefacts mistaken for disease, leading to incorrect diagnoses or misinterpretation of research findings. Proper handling of muscle biopsies is critical for accurate histopathological and mitochondrial analysis. It is essential to preserve the entire tissue, especially for small needle biopsies. While most research focuses on mitochondrial analysis in cells, there are few studies on whole tissue samples. This study aimed to provide an effective methodological workflow to improve cryopreservation techniques for human and rodent muscle biopsies and create a reliable method for mitochondrial analysis in muscle tissues. Methods: Human muscle samples were preserved with different concentrations of formaldehyde after freezing with liquid nitrogen to study the effects of freeze–thaw cycles. We compared the edge and belly of muscle samples embedded in Optimal Cutting Temperature compound (OCT) to see how OCT affects ice crystal formation. Rat muscle biopsies were frozen using six different methods, using liquid nitrogen and precooled isopentane as freezing media. Each medium involved direct immersion, OCT dip before immersion, and placement in histocassettes before immersion. Effectiveness of these methods was evaluated using histological and immunohistochemical staining. Mitochondrial analysis in type I and II myofibres was attempted by employing the Trainable Weka Segmentation plugin using Fiji. Results: Histologically stained human tissue sections showed that freeze–thaw and formaldehyde fixation led to freezing artefacts, disrupted endomysium, and widely spaced cells. Quantitative differences in ice crystal artefacts between edge and belly of rat whole muscle samples demonstrated effects of OCT in crystal formation. Histological and immunohistochemical staining of sections from rat muscle biopsies frozen in six different cryopreservation techniques revealed that only isopentane/histocassette combination preserved tissue integrity in both core and periphery of tissue sections. Moreover, an optimised Fiji workflow enabled accurate quantification and mapping of mitochondrial networks. Discussion: The isopentane/histocassette combination is an effective cryopreservation method, ensuring artefact-free preservation of both core and periphery of tissue sections. Our workflow utilising Trainable Weka Segmentation plugin provides a reliable method for mitochondrial analysis in skeletal muscle tissues, facilitating future studies in muscle research. [ABSTRACT FROM AUTHOR]

Background: Astrephomene is an interesting green algal genus that, together with Volvox, shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two species of Astrephomene were described. However, maintenance of culture strains of Astrephomene requires rapid inoculation of living cultures, and cryopreserved culture strains have not been established in public culture collections. Results: To establish cryopreserved culture strains of two species of Astrephomene, conditions for cryopreservation of the two species were investigated using immature and mature vegetative colonies and two cryoprotectants: N,N-dimethylformamide (DMF) and hydroxyacetone (HA). Rates of cell survival of the A. gubernaculifera or A. perforata strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3 and 6%) of DMF and HA and two types of colonies: immature colonies (small colonies newly released from the parent) and mature colonies (large colonies just before daughter colony formation). The highest rate of survival [11 ± 13% (0.36–33%) by the most probable number (MPN) method] of A. gubernaculifera strain NIES-4017 (established in 2014) was obtained when culture samples of immature colonies were subjected to cryogenic treatment with 6% DMF. In contrast, culture samples of mature colonies subjected to 3% HA cryogenic treatment showed the highest "MPN survival" [5.5 ± 5.9% (0.12–12%)] in A. perforata. Using the optimized cryopreservation conditions for each species, survival after freezing in liquid nitrogen was examined for six other strains of A. gubernaculifera (established from 1962 to 1981) and another A. perforata strain maintained in the Microbial Culture Collection at the National Institute for Environmental Studies (MCC-NIES). We obtained ≥0.1% MPN survival of the A. perforata strain. However, only two of the six strains of A. gubernaculifera showed ≥0.1% MPN survival. By using the optimal cryopreserved conditions obtained for each species, five cryopreserved strains of two species of Astrephomene were established and deposited in the MCC-NIES. Conclusions: The optimal cryopreservation conditions differed between the two species of Astrephomene. Cryopreservation of long-term-maintained strains of A. gubernaculifera may be difficult; further studies of cryopreservation of these strains are needed. [ABSTRACT FROM AUTHOR]

Aims: We analysed the survival, complications, and function of frozen autograft augmented with intramedullary cement and bridging plates fixation for intercalary bone defect reconstruction in primary bone sarcomas. Patients and Methods: A retrospective cohort study was conducted on 72 patients with primary bone sarcomas (34 males, 38 females) between January 2016 and June 2023. The average age was 22.0 ± 13.6 years (6 to 61 years) and the pathological type included osteosarcoma (55), followed by adamantinoma (5), Ewing's sarcoma (4), undifferentiated pleomorphic sarcoma (4), chondrosarcoma (3), and malignant tenosynovial giant cell tumor (1). The oncological outcomes included local control, metastasis, progression-free survival and overall survival. The functional outcomes were evaluated by the Musculoskeletal Tumor Society Score (MSTS-93), the Toronto Extremity Salvage Score (TESS), and the motion of the joint. Results: The mean follow-up time was 50.0 ± 27.4 months (12 to 99 months). 10 patients died of the disease, 9 patients were alive with disease and 53 patients were alive with no evidence of disease. The average 5-year overall survival of autograft was 85.8% (95% CI, 72.1-93.1%). The average MSTS-93 score was 96% (67–100%) and the average TESS score was 98% (74–100%). Twenty-four patients (33.3%) had at least one complication in the follow-up period. The most common complications were nonunion (9.7%, 7/72) and local recurrence (9.7%, 7/72), followed by leg length discrepancy (6.9%, 5/72), infection (5.6%, 4/72), implant failure (4.2%, 3/72), delayed union (2.8%, 2/72), and graft fractures (1.4%, 1/72). Tumor site was an independent risk factor for bone nonunion (OR, 22.23; p = 0.006). Conclusions: We presented a large technique series for preventing autograft-related complications (especially for autograft fractures) of intercalary frozen autograft reconstruction. This method showed promising functional outcomes and provided durable reconstruction. Level of evidence: level IV therapeutic study. [ABSTRACT FROM AUTHOR]

Background: Ovarian cryopreservation is a promising technique despite being hindered by damage from freezing and thawing. Melatonin can mitigate this outcome. Objective: This study aimed to evaluate the effect of melatonin on the follicular dynamics of ovarian tissue in a cryopreserved cell culture. Methods: Three-month-old adult female Wistar rats (n = 24) weighing approximately 250 g were oophorectomized and divided into two groups (n = 12): the control group (CG) and the melatonin group (MG). In the CG, slow cryopreservation was performed according to the standard protocol with Medium M2 and dimethyl sulfoxide (DMSO). In MG, melatonin diluted in ethyl alcohol vehicle at a concentration of 0.1 μm was added to the culture medium. In both groups, the ovaries were cryopreserved by slow freezing and kept in liquid nitrogen for 24 h. Subsequently, after thawing, the ovaries were reimplanted in the retroperitoneum, one on each side of the great vessels (inferior vena cava and aorta). After 30 days, the animals were euthanized during the diestrus phase; then, the grafts were removed and processed for histomorphometric and immunohistochemical analyses, whereas the blood was subjected to biochemical analysis. Student's t test was used to assess the difference between the groups. Results: The FSH levels in MG (83.79 ± 32.37) were lower than those in CG (120.52 ± 36.59), p = 0.03. The FSH/AMH ratios were also lower in MG (3.53 ± 1.13) than in CG (6.52 ± 2.85), p = 0.001. The SOD2 immunoexpression was higher in MG than in CG regarding all parameters except for the degenerated follicles (follicular cells and internal thecal cells): CG (16.80 ± 4.80 [13.36–20.24]) and MG (14.91 ± 4.04 [12.01–17.79]) p = 0.351. Statistically, the difference in intact follicles (theca + interstitium) between CG (6.60 ± 2.59 [4.75–8.45]) and MG (9.31 ± 3.09 [7.09–11.51]) was significant (p = 0.049), with a small difference in the expression of regular antral follicles. Conclusions: Melatonin can improve the quality of cryopreserved tissues, as evidenced in this study, and the evaluation of cryopreserved ovarian grafts, as shown in the melatonin group with better hormonal parameters and greater immunohistochemical expression of the SOD2 antioxidant. Thus, damage is reduced during cryopreservation and transplantation is improved. [ABSTRACT FROM AUTHOR]

Background: Cachexia a multifactorial syndrome is a common sequala in patients with cancer. It varies from 42 to 80% depending upon the oncological stage and is directly responsible for 30% of deaths in these patients. Previous research from our laboratory demonstrated that peritoneal ovarian cancer generated in NSG mice resulted in skeletal and cardiac muscle atrophy - leading to loss of skeletal muscle mass and strength, and cardiac dysfunction (cachexia). Treatment of mice bearing i.p. tumors with withaferin A (WFA) showed reversal of skeletal muscle and cardiac cachexia. The present study is focused on determining effects of peritoneal ovarian tumors on kidney damage and effects of WFA treatment on ameliorating kidney damage. Methods: We generated intraperitoneal ovarian cancer by injecting female NSG mice with ovarian cancer cell line (A2780). After one week of injecting cancer cells, mice were treated with WFA (4 mg/kg) every third day, for three weeks. After 4 weeks of injection of cancer cells, the mice were sacrificed and various tissues including kidney and blood were collected, snap-frozen in liquid nitrogen, and stored at -800C. The presence of kidney biomarker creatinine, was measured in the plasma by an ELISA. The mRNA was isolated from mouse kidneys and was used to examine the expression levels of signaling proteins, inflammatory cytokines, and genes responsible for inducing cachexia (IL-1β, IL-6, TNF-α, TGF-β, GDF-15, and MYD88). Results: Our results showed a significant increase in levels of expression of inflammatory cytokine IL-1 β (p < 0.01), IL-6 (p < 0.001), TNF-α (p < 0.001), and other related genes including TRAF6 (p < 0.01), MYD88 (p < 0.01), and GDF-15 (p = 0.005) in tumor-bearing mice compared to controls. Treatment of mice bearing tumors with WFA attenuated the increase in expression of each gene. In addition, our results showed a significant increase in creatinine levels in circulation in tumor-bearing mice compared to control mice. Treatment of tumor-bearing mice with WFA resulted in a significant decrease in plasma creatinine levels compared to tumor-bearing mice. Conclusions: Our results conclude that ovarian tumors in NSG mice caused kidney damage and renal dysfunction, which was effectively ameliorated by WFA treatment, suggesting a protective effect of WFA on kidney injury induced by ovarian cancer. [ABSTRACT FROM AUTHOR]

Objective: Cutaneous Leishmaniasis (CL) is one of the highly prevalent endemic diseases in the Middle East. The disease is a complex skin infection imposing a heavy burden on many developing countries. This study aimed to evaluate the impact of adding oral fluconazole to topical cryotherapy on the treatment efficacy and time to achieve complete recovery of CL lesions. Method: This triple-blind randomized clinical trial included 52 participants with CL. Participants were allocated to receive either weekly cryotherapy with liquid nitrogen and oral fluconazole at a dose of 6 mg/kg daily at a maximum of 400 mg for 6 weeks as the interventional arm or weekly cryotherapy with liquid nitrogen plus the placebo for the same period of 6 weeks as the control arm. Results: Fifty-two eligible participants enrolled the study, with a CL lesion count of 1 to 8 (mean 1.96), and served as the interventional (n = 28) and control (n = 24) arms. The trend of the mean surface area of the lesions was significantly decreasing in both arms (P < 0.001), with no statistically significant difference between arms (P = 0.133) or all assessed time point pairwise comparisons (P > 0.05). There was no significant difference between the treatment arms in terms of the end-point recovery status (P = 0.491) or the frequency of post-treatment secretion (P = 0.437). No adverse effect was observed. Conclusion: Despite a slightly higher reduction in the lesion surface in the cryotherapy and fluconazole treatment arm, the addition of fluconazole did not provide statistically significant therapeutic value to cryotherapy in the treatment of CL. However, with adjustment for the initial lesion size, the efficacy of the regimen in the interventional arm was more pronounced, though it was still insignificant. [ABSTRACT FROM AUTHOR]

The clinical application of cell therapies is becoming increasingly important for the treatment of cancer, congenital immune deficiencies, and hemoglobinopathies. These therapies have been primarily manufactured and used at academic medical centers. However, cell therapies are now increasingly being produced in centralized manufacturing facilities and shipped to medical centers for administration. Typically, these cell therapies are produced from a patient’s own cells, which are the critical starting material. For these therapies to achieve their full potential, more medical centers must develop the infrastructure to collect, label, cryopreserve, test, and ship these cells to the centralized laboratories where these cell therapies are manufactured. Medical centers must also develop systems to receive, store, and infuse the finished cell therapy products. Since most cell therapies are cryopreserved for shipment and storage, medical centers using these therapies will require access to liquid nitrogen product storage tanks and develop procedures to thaw cell therapies. These services could be provided by the hospital pharmacy or transfusion service, but the latter is likely most appropriate. Another barrier to implementing these services is the variability among providers of these cell therapies in the processes related to handling cell therapies. The provision of these services by medical centers would be facilitated by establishing a national coordinating center and a network of apheresis centers to collect and cryopreserve the cells needed to begin the manufacturing process and cell therapy laboratories to store and issue the cells. In addition to organizing cell collections, the coordinating center could establish uniform practices for collecting, labeling, shipping, receiving, thawing, and infusing the cell therapy. [ABSTRACT FROM AUTHOR]

Background: Cryopreservation makes it possible to preserve plant biodiversity for thousands of years in ex situ storage. The stepwise dehydration method is a simple and versatile cryopreservation technique based on the vitrification phenomenon. However, the commonly used dimethyl sulfoxide (DMSO) in this cryopreservation technique is considered harmful for plant material, thus alternative methods are needed to be applied. Results: In this study, the possibility of cryopreservation of embryogenic tissues (ETs) of Abies alba x A. numidica and Pinus nigra was investigated. Before freezing, ETs were partially dehydrated in the presence of increasing concentrations of sucrose (from 0.25 to 1.0 M) for 7 days, followed by desiccation of the tissues over silica gel for 2 and 2.5 h, respectively. After these pretreatments, the plant material was frozen in liquid nitrogen (LN; –196 °C). For both coniferous trees the ET survival rate was high and reached 84.4% for A. alba x A. numidica (28 days) and 86.7% for P. nigra (35 days) after recovery of the tissues from liquid nitrogen (LN). The regenerated tissue of A. alba x A. numidica was characterized by more intense growth after storage in LN compared to tissue that had not been cryopreserved (control). The tissue of this tree also undertook relatively rapid growth after thawing from LN. In turn, the ET growth of P. nigra was significantly lower after thawing compared to the other treatment. Conclusions: The present study demonstrated, that the stepwise dehydration method could be successfully applied to the cryostorage of ETs of both studied trees. To the best of our knowledge, this is the first report on ET cryopreservation based on this method for Abies and Pinus genus representatives, which may be the alternative way for efficient, long-term preservation of germplasm in LN. [ABSTRACT FROM AUTHOR]

Background: With the rapid development of single-cell sequencing technology, histological studies are no longer limited to conventional homogenized tissues. Laser microdissection enables the accurate isolation of specific tissues or cells, and when combined with next-generation sequencing, it can reveal important biological processes at the cellular level. However, traditional laser microdissection techniques have often been complicated and time-consuming, and the quality of the RNA extracted from the collected samples has been inconsistent, limiting follow-up studies. Therefore, an improved, simple, and efficient laser microdissection method is urgently needed. Results: We omitted the sample fixation and cryoprotectant addition steps. Instead, fresh samples were embedded in Optimal Cutting Temperature medium within 1.5 ml centrifuge tube caps, rapidly frozen with liquid nitrogen, and immediately subjected to cryosectioning. A series of section thicknesses of citrus rind were tested for RNA extraction, which showed that 18 μm thickness yielded the highest quality RNA. By shortening the dehydration time to one minute per ethanol gradient and omitting the tissue clearing step, the resulting efficient dehydration and preserved morphology ensured high-quality RNA extraction. We also propose a set of laser microdissection parameters by adjusting the laser power to optimal values, reducing the aperture size, and lowering the pulse frequency. Both the epidermal and subepidermal cells from the citrus rind were collected, and RNA extraction was completed within nine hours. Using this efficient method, the transcriptome sequencing of the isolated tissues generated high-quality data with average Q30 values and mapping rates exceeding 91%. Moreover, the transcriptome analysis revealed significant differences between the cell layers, further confirming the effectiveness of our isolation approach. Conclusions: We developed a simple and rapid laser microdissection method and demonstrated its effectiveness through a study based on citrus rind, from which we generated high-quality transcriptomic data. This fast and efficient method of cell isolation, combined with transcriptome sequencing not only contributes to precise histological studies at the cellular level in citrus but also provides a promising approach for cell-specific transcriptome analysis in a broader range of other plant tissues. [ABSTRACT FROM AUTHOR]

Background: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10−9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10−9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification). [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the effectiveness of combining a customized mold with frozen conventional clamps against other freezing and non-freezing methods. Methods: Forty-five porcine and 45 chicken tendons were evenly divided into five groups (n = 9 + 9/group): control group, non-freezing with gauze placed between tendon and clamp (gauze), non-freezing with suture fixation at tendon ends (suture), freezing with dry ice pocket placed at the clamps (pocket), and freezing using a templated liquid nitrogen clamp with a customized mold (mold). Tension tests were used to measure failure modes and loads. Result: Slippage and avulsion were observed in non-freezing groups with significantly lower failure loads compared to freezing methods. With freezing, rupture occurred near the central point only in the mold group. The failure loads for porcine tendons in the mold group were higher (2121.651 ± 73.101 N) than the pocket group (1746.337 ± 68.849 N). The failure loads of chicken tendons in the mold (243.552 ± 15.881 N) and pocket groups (260.647 ± 22.161 N) were not statistically different. Conclusion: Freezing clamps represent the better choice for soft tissue clamping. The customized mold method could improve gripping effectiveness. [ABSTRACT FROM AUTHOR]

Background: Boar sperm are highly susceptible to specific conditions during cryopreservation, leading to a significant decrease in their fertilizing potential due to damage to their membranes. Camellia oil, known for its fatty acids with antioxidant and biological properties, has not been previously explored for the cryopreservation of boar semen. This study aimed to examine the effects of camellia oil on post-thawed boar sperm quality. Boar semen ejaculates (n = 9) were collected and divided into six equal aliquots based on camellia oil concentrations (0, 0.5, 1, 1.5, 2 and 2.5% v/v) in the freezing extender. Semen samples were processed and cryopreserved using the liquid nitrogen vapor method. Thereafter, frozen semen samples were thawed at 50 °C for 12 s and evaluated for sperm morphology by scanning electron microscope, sperm motility using a computer-assisted sperm analyzer, sperm viability, acrosome integrity, mitochondrial function, MDA level and total antioxidant capacity. Results: The results demonstrated that the supplementation of 1.5% (v/v) camellia oil showed superior post-thaw sperm qualities such as improved sperm morphology, motility, acrosome integrity and mitochondrial function by 14.3%, 14.3% and 11.7%, respectively, when compared to the control group. Camellia oil at a concentration of 1.5% (v/v) showed the lowest level of MDA (18.3 ± 2.1 µmol/L) compared to the other groups. Conclusions: In conclusion, adding 1.5% (v/v) camellia oil in the freezing extender reduced the oxidative damage associated with cryopreservation and resulted in a higher post-thawed sperm quality. [ABSTRACT FROM AUTHOR]

Objective: This study was aimed to use a digital design of 3D-printing technology to create a surgical navigation template. At the same time, biphasic calcium phosphate (BCP) was applied to treat osteonecrosis of the femoral head (ONFH) in animal models, based on accurate positioning of necrotic lesions in the navigation templates and observation of its therapeutic effect. Methods: Fifteen healthy adult male and female beagle dogs weighing 20 + 2 kg were randomly divided into three groups (n = 5) after establishing a model of ONFH using the liquid nitrogen freezing method. Each model underwent necrotic lesion creation and BPC implantations on one side of the femoral head and only necrotic lesion creation on the other side of the femoral head. Each group underwent CT examination, gross observation, histological examination and immunohistochemical staining at 6 weeks, 12 weeks and 18 weeks postoperatively. Results: At weeks 6, 12, and 18, CT and gross examination showed that the necrotic area in the experimental group was basically intact and had been completely raised by BCP material. In the control group, there were signs of bone repair in the femoral head, but there were still large bone defects and cavities. At week 18, extensive collapse of the cartilage surface was observed. Through histological examination, in the experimental group at 12 and 18 weeks, a large number of new and reconstructed bone trabeculae containing a large amount of collagen fibres were observed (P < 0.05), while in the control group, there was extensive necrosis of the bone trabeculae without cellular structural areas. Immunohistochemical examination observation: A large number of CD31-positive cells were observed in the experimental group at 6 weeks, gradually decreasing at 12 and 18 weeks (P < 0.05), while a small number of CD31-positive cells were observed in the control group at 18 weeks. Conclusion: The 3D-printed navigation template can accurately locate ONFH lesions. Implantation of BCP material can effectively play a supporting role, prevent the collapse of the loading surface, and induce bone formation and angiogenesis to some extent. [ABSTRACT FROM AUTHOR]

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Background: Dermatophagoides farinae proteins (DFPs) are abundantly expressed in D. farinae; however, their functions remain unknown. Our previous transcriptome sequencing analyses revealed that the basal expression of DFP1 and DFP2 in D. farinae was high and, more importantly, upregulated under temperature stress. Therefore, DFPs were speculated to exert a temperature stress response function. Results: Real-time quantitative polymerase chain reaction detection revealed that both DFP1 and DFP2 were significantly upregulated under temperature stress. Particularly, DFP1 was upregulated under cold stress. Electrophoresis of D. farinae total proteins revealed an increased abundance of DFP1 and DFP2 (40–55 kDa bands) under temperature stress, which was corroborated by the mass spectrometry results. After silencing DFP1 and DFP2 further, temperature stress led to decreases in gene expression and survival rates. Moreover, DFP1 was identified as the upstream regulator of DFP2. Conclusion: This study highlights the temperature stress response functions of DFP1 and DFP2 at the mRNA and protein levels. These results provide important insights for applying DFP1 and DFP2 as potential target genes for the molecular prevention and control of D. farinae to prevent allergic diseases. The newly established methods provide methodological guidance for the study of genes with unknown functions in mites. [ABSTRACT FROM AUTHOR]

Over the two last decades, whole-body cryotherapy/cryostimulation (WBC) has emerged as an exciting non-pharmacological treatment influencing inflammatory events at a cellular and physiological level, which can result in improved sleep quality, faster neuromuscular recovery after high-intensity exercise, and chronic pain relief for patients suffering different types of diseases (fibromyalgia, rheumatism, arthritis). Some evidence even suggests that WBC has benefits on mental health (depression, anxiety disorders) and cognitive functions in both adults and older adults, due to increased circulating BDNF levels. Recently, some safety concerns have been expressed by influential public health authorities (e.g., FDA, INSERM) based on reports from patients who developed adverse events upon or following WBC treatment. However, part of the data used to support these claims involved individuals whose entire body (except head) was exposed to extreme cold vaporized liquid nitrogen while standing in a narrow bathtub. Such a procedure is known as partial-body cryotherapy (PBC), and is often erroneously mistaken to be whole-body cryotherapy. Although having similarities in terms of naming and pursued aims, these two approaches are fundamentally different. The present article reviews the available literature on the main safety concerns associated with the use of true whole-body cryotherapy. English- and French-language reports of empirical studies including case reports, case series, and randomized controlled trials (RCTs) were identified through searches of PubMed, Scopus, Cochrane, and Web of Science electronic databases. Five case reports and two RCTs were included for a total of 16 documented adverse events (AEs). A critical in-depth evaluation of these AEs (type, severity, context of onset, participant's medical background, follow-up) is proposed and used to illustrate that WBC-related safety risks are within acceptable limits and can be proactively prevented by adhering to existing recommendations, contraindications, and commonsense guidelines. [ABSTRACT FROM AUTHOR]

Background: Extra-adrenal myelolipoma is an unusual entity, and endobronchial myelolipoma is rarer, which is often ignored by clinicians, delaying the disease and affecting the prognosis. Case presentation: A 71-year-old man with a history of chronic obstructive pulmonary disease (COPD) and type 2 diabetes mellitus, with recurrent fever, cough, and expectoration for more than 2 weeks experienced relief in cough, phlegm reduction, and glycemic control with anti-inflammatory treatment. Further examination revealed that new growths obstructing all lobar bronchi impaired flexible bronchoscope entry. In order to relieve the patient's symptoms, under general anesthesia, we performed liquid nitrogen cryobiopsy at multiple bronchial openings, and then used argon plasma coagulation (APC) to achieve hemostasis. The pathological diagnosis was bronchial myelolipoma. The largest volume of the resected tissue was a mass measuring 0.6 cm × 0.4 cm × 0.3 cm at the bronchial opening of the upper lobe of the left lung. The patient's condition was stable and the symptoms were partially relieved after surgery. No recurrence was observed during the 12-month follow-up, although the long-term treatment efficacy is unknown. Conclusion: Pathological biopsy is key to the diagnosis of endobronchial myelolipoma, and the development of the endobronchial myelolipomas may have been associated with long-term poor control of steroid levels in this patient. [ABSTRACT FROM AUTHOR]

Objectives: Explore the therapeutic role of human umbilical mesenchymal stem cells (hUMSCs) transplantation for regeneration of ECM components and restoration of follicular development in mice. Background: The extracellular matrix (ECM) is crucial to maintain ovary function and regulate follicular development, as it participates in important cell signaling and provides physical support to the cells. However, it is unknown how hUMSCs affect the expression of ECM-related genes in ovaries treated with cyclophosphamide (CTX) and busulfan (BUS). Methods: In the present study, we used 64 six- to eight-week-old ICR female mice to established mouse model. The mice were randomly divided into four groups (n = 16/group): control, POI, POI + hUMSCs, and POI + PBS group. The premature ovarian insufficiency (POI) mouse model was established by intraperitoneal injection of CTX and BUS for 7days, then, hUMSCs or PBS were respectively injected via the tail vein in POI + hUMSCs or POI + PBS group. Another 7days after injection, the mice were sacrificed to harvest the ovary tissue. The ovaries were immediately frozen with liquid nitrogen or fixed with 4% PFA for subsequent experiments. To screen differentially expressed genes (DEGs), we performed transcriptome sequencing of ovaries. Thereafter, a Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the related biological functions. Retrieval of interacting genes for ECM-related DEGs was performed using the function of STRINGdb (version 2.6.5) to evaluate potential protein-protein interaction (PPI) networks. Furthermore, qRT-PCR and IHC were performed to assess the differential expression of selected DEGs in control, damaged, hUMSCs-transplanted and non-transplanted ovaries. Results: Chemotherapy caused mouse ovarian follicular reserve depletion, and hUMSCs transplantation partially restored follicular development. Our results revealed that ECM-receptor interaction and ECM organization were both downregulated in the damaged ovaries. Further investigation showed that ECM-related genes were downregulated in the CTX and BUS treatment group and partially rescued in hUMSCs injection group but not in the PBS group. qRT-PCR and IHC verified the results: collagen IV and laminin gamma 3 were both expressed around follicle regions in normal ovaries, chemotherapy treatment disrupted their expression, and hUMSCs transplantation rescued their localization and expression to some extent. Conclusion: Our data demonstrated that ECM-related genes participate in the regulation of ovarian reserve, hUMSCs treatment rescued abnormal expression and localization of collagen IV and laminin gamma 3 in the damaged ovaries. The results suggest that hUMSCs transplantation can maintain ECM-stable microenvironments, which is beneficial to follicular development. [ABSTRACT FROM AUTHOR]

Background: Cryopreservation of embryos plays a major role in the in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. However, the storage condition of the cryopreserved embryo can change temporarily due to repeated retrieval of the embryo from the liquid nitrogen (LN2) tank during the practical application during cryopreservation. Whether the implantation potential of a cryopreserved embryo will be damaged when the cane containing it is temporarily exposed to air due to the transfer between the LN2 tank and LN2 container is yet to be elucidated. Also, whether the exposed-to-air frequency (EAF) of cryopreserved embryos influences the clinical outcomes is unclear. Objective: To investigate whether the EAF of cryopreserved embryo affects the clinical outcomes of vitrified-warmed embryo transfer. Methods: A total of 9200 vitrified-warmed embryo transfer cycles were included in this study. All cycles were divided into five groups according to different EAFs (2, 4, 6, 8, or ≥ 10). Post-warming survival rates and clinical outcomes, including implantation, clinical pregnancy and live birth rates were investigated. Kruskal–Wallis test and Pearson's chi-squared tests were used to compare the patient characteristics and clinical outcomes among the five groups. Furthermore, multivariate logistic regression analyses were conducted to investigate the association between EAF and clinical outcomes. Results: No significant differences were observed in the positive HCG rate, implantation rate and live birth rate (P > 0.05) among five EAF groups with respect to D3 embryo, D5 blastocyst and D6 blastocyst. Post-warmed survival rate of D3 embryos (P = 0.015) differed significantly among the five EAF groups, but it was not EAF-dependent. Although clinical pregnancy was different among the five groups with respect to D5 blastocyst (P = 0.042), multivariate logistic regression analysis adjusted for confounding variables suggested that EAF did not adversely affect clinical pregnancy or live birth. Conclusion: These findings indicated that human vitrified embryos in the open system could be repeatedly retrieved from the LN2 tank without affecting the implantation potential of the embryo. [ABSTRACT FROM AUTHOR]

Background: Hyperlipidemia, a heterogeneous group of disorders characterized by elevated plasma lipids in the blood, causes severe health problems, leading to fatty liver disease and nonalcoholic fatty liver disease. Thymoquinone, the major active chemical component of Nigella sativa, reportedly exerts a vast array of biological effects. Various studies have reported that Thymoquinone protects against liver injury. Aims: The aim of this study was to investigate the possible protective effects of Thymoquinone against liver injury in hyperlipidemia-induced LDL-R−/− mice. Methods: Eight-week-old male LDL-R−/− mice were randomly divided into three groups: a control group fed a normal diet and two groups fed a high-cholesterol diet or high-cholesterol diet mixed with Thymoquinone. All groups were fed different diets for 8 weeks. Blood samples were obtained from the inferior vena cava and collected in serum tubes. The samples were then stored at − 80 °C until used. Longitudinal sections of liver tissues were fixed in 10% formalin and then embedded in paraffin for histological evaluation. The remainder of the liver tissues were snap-frozen in liquid nitrogen for reverse transcription-polymerase chain reaction or western blotting. Results: Our results demonstrated that Thymoquinone administration significantly reduced liver histological alterations by hyperlipidemia. Thymoquinone mitigated hyperlipidemia-induced liver injury as indicated by the suppression of metabolic characteristics, liver biochemical parameters, pyroptosis indicators, a macrophage marker, and the phosphatidylinositide 3-kinase signaling pathway. Conclusions: Thymoquinone is a potential therapeutic agent for hyperlipidemia-induced liver injury. [ABSTRACT FROM AUTHOR]

Background: Stable isotope labeling is a non-invasive, sensitive means of monitoring metabolic flux in plants. The most physiologically meaningful information is obtained from experiments that take advantage of the natural photosynthetic carbon assimilation pathway to introduce a traceable marker with minimal effects on the physiology of the organism. The fundamental substrate in isotopic labeling experiments is 13CO2, which can reveal the earliest events in carbon assimilation and realistically portray downstream metabolism when administered under conditions suitable for making kinetic inferences. Efforts to improve the accuracy and resolution of whole plant labeling techniques have focused on improvements in environmental control, air flow characteristics, and harvesting methods. Results: Here we present a dynamic flow cuvette designed for single Arabidopsis thaliana labeling experiments. We have also verified its suitability for labeling Nicotiana benthamiana and essential oils in Pelargonium graveolens. Complete plans for fabrication of this device are included. The design includes three important innovations. First, uniform, circular air flow over the rosette surface is accomplished by a fan and deflector that creates a mini-cyclone effect within the chamber interior. Second, a network of circulating canals connected to a water bath provides temperature control to within ± 0.1 ºC under variable irradiance, humidity, and air flow conditions. When photosynthetically active radiation (PAR) was varied over a range of 1000 μEinsteins m−2 s−1 with no adjustment to the external temperature control system, the abaxial leaf temperature changed by < 3 ºC/1000 PAR. Third, the device is fully compatible with liquid nitrogen quenching of metabolic activity without perturbation of the light environment. For short labeling experiments (< 10 s), the most critical variable is the half-life (t1/2) of the atmosphere within the chamber, which determines the maximum resolution of the labeling system. Using an infrared gas analyzer, we monitored the atmospheric half-life during the transition from 12CO2 to 13CO2 air at different flow rates and determined that 3.5 L min−1 is the optimal flow rate to initiate labeling (t1/2 ~ 5 s). Under these conditions, we observed linear incorporation of 13C into triose phosphate with labeling times as short as 5 s. Conclusions: Advances in our ability to conduct short term labeling experiments are critical to understanding of the rates and control of the earliest steps in plant metabolism. Precise kinetic measurements in whole plants using 13CO2 inform metabolic models and reveal control points that can be exploited in agricultural or biotechnological contexts. The dynamic labeling cuvette presented here is suitable for studying early events in carbon assimilation and provides high resolution kinetic data for studies of metabolism in intact plants under physiologically realistic scenarios. [ABSTRACT FROM AUTHOR]

Objective: The need for innovative techniques to preserve microbiota for extended periods, while maintaining the species composition and quantitative balance of the bacterial community, is becoming increasingly important. To address this need, we propose an efficient approach to cryopreserve human gut microbiota using a two-component cryoprotective composition comprising fetal bovine serum (FBS) and 5% dimethyl sulfoxide (DMSO). Fetal serum is a commonly utilized component in the freezing media for eukaryotic cells, however, its effects on prokaryotic cells have not been extensively researched. Results: In our study, we demonstrated the high efficiency of using a two-component cryoprotective medium, FBS + 5% DMSO, for cryopreservation of human gut microbiota using three different methods. According to the obtained results, the intact donor microbiota was preserved at a level of 85 ± 4% of the initial composition based on fluorescent analysis using the LIVE/DEAD test. No differences in survival were observed when comparing with pure DMSO and FBS media. The photometric measurement method for growth of aerobic bacteria (A. johnsoni), facultative anaerobes (E. coli, E. faecalis), microaerophilic (L. plantarum), and obligate anaerobic bacterial cultures (E. barkeri, B. breve) also demonstrated high viability rates of 94–98% in the two-component protective medium, reaching intact control levels. However, for anaerobic microflora representatives, serum proved to be a more suitable cryoprotectant. Also, we demonstrated that using cryoprotective media with 50–75% FBS content is enough to preserve a significant level of bacterial cell viability, from an economic standpoint. [ABSTRACT FROM AUTHOR]

Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function. [ABSTRACT FROM AUTHOR]

Background: High temperature seriously limits the annual production of fresh cut lilies, which is one of the four major cut flowers in the global cut flower market. There were few transcriptomes focused on the gene expression of lilies under heat stress. In order to reveal the potential heat response patterns in bulbous plants and provide important genes for further genetic engineering techniques to improve thermotolerance of lily, RNA sequencing of lilies under heat treatments were conducted. Results: In this study, seedlings of Lilium longiflorum 'White Heaven' were heat-treated at 37 °C for different lengths of time (0 h, 0.5 h, 1 h, 3 h, 6 h, and 12 h with a 12 h-light/12 h-dark cycle). The leaves of these lily seedlings were immediately collected after heat treatments and quickly put into liquid nitrogen for RNA sequencing. 109,364,486–171,487,430 clean reads and 55,044 unigenes including 21,608 differentially expressed genes (DEGs) (fold change ≥2) were obtained after heat treatment. The number of DEGs increased sharply during the heat treatments of 0.5 h–1 h and 1 h–3 h compared to that of other periods. Genes of the heat stress transcription factor (HSF) family and the small heat shock proteins (small HSPs, also known as HSP20) family responded to heat stress early and quickly. Compared to that of the calcium signal and hormone pathways, DEGs of the HSF-HSP pathway and reactive oxygen species (ROS) pathway were significantly and highly induced. Moreover, they had the similar expression pattern in response to heat stress. Small HSPs family genes were the major components in the 50 most highly induced genes at each heat stress treatment and involved in ROS pathway in the rapid response to heat stress. Furthermore, the barley stripe mosaic virus induced gene silencing (BSMV-VIGS) of LlHsfA2 caused a significantly reduced thermotolerance phenotype in Lilium longiflorum 'White Heaven', meanwhile decreasing the expression of small HSPs family genes and increasing the ROS scavenging enzyme ascorbate peroxidase (APX) genes, indicating the potential interplay between these two pathways. Conclusions: Based on our transcriptomic analysis, we provide a new finding that small HSPs play important roles in crosstalk between HSF-HSP and ROS pathways in heat stress response of lily, which also supply the groundwork for understanding the mechanism of heat stress in bulbous plants. [ABSTRACT FROM AUTHOR]

Background: The application of ribo nucleic acids for molecular studies requires high integrity and quality of extracted total RNA samples. In addition, the need to transfer RNA samples at room temperature without special treatments such as ice and liquid nitrogen storage according to international transport laws highlights the importance of low cost alternative methods such as RNA air-drying, lyophilisation and transportable agents. In this study, the quality and quantity of air-dried RNA samples from leaf, petiole and bark tissues of different olive genotypes using several RNA extraction methods were compared with lyophilized ground leaves and RNAlater-stored tissue samples before precipitation. The quality of RNA and prepared libraries were checked by several techniques including agarose and polyacrylamide gel electrophoresis, Agilent quality control, RT-PCR amplification of housekeeping and viral genes and high throughput sequencing. Results: Although RNA value varied amongst cultivars, RNA extraction with TRIzol™ Reagent in fresh extractions and samples stored in RNAlater before RNA extraction resulted in 455.26 ng/µL and 63.46 ng/µL (mean value of cultivars) as the highest RNA concentration averages, respectively. RNA samples extracted by TRIzol™ Reagents and stored for a short term at – 80 °C before air-drying showed the third highest concentration (44.87 ng/µL). The synthesized cDNAs quality for PCR amplification of housekeeping genes (Rbc 1 and Nad 5) and partial genomes of Arabis mosaic virus and Cucumber mosaic virus showed satisfactory results in RNA samples extracted by TRIzol™ Reagents despite its variation amongst cultivars. Conclusions: Considering the difficulties in the extraction of high quality and quantity RNA in olive for molecular analyses, this study demonstrated that RNA extraction method based on TRIzol™ Reagent can be considered for virobiome studies of both fresh and air-dried samples. [ABSTRACT FROM AUTHOR]

Background: Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use.Methods: Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells.Results: After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells.Conclusion: We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products. [ABSTRACT FROM AUTHOR]

Background: High-resolution respirometry (HRR) of human biopsies can provide useful metabolic, diagnostic, and mechanistic insights for clinical research and comparative medical studies. Fresh tissues analysis offers the potential best condition, the drawback being the need to use them shortly after dissection for mitochondrial respiratory experiments. The development of effective long-term storage protocols for biopsies that allow the assessment of key Electron Transport System (ETS) parameters at later stages is thus a major need. Methods: We optimised a cryopreservation protocol that preserves mitochondrial membranes intactness, otherwise affected by direct tissue freezing. The protocol is based on a gradual freezing step from on-ice to liquid nitrogen and − 80 °C storage using a specific DMSO-based buffer. Results: Placenta is a suitable tissue to design and test the effectiveness of long-term storage protocols being metabolically active foetal tissue with mitochondrial dysfunctions contributing to placental disease and gestational disorders. Here we designed and tested the effectiveness of the cryopreservation protocol using human placenta biopsies; we measured the ETS activity by HRR of placenta specimens comparing fresh, cryopreserved, and snap frozen conditions. Conclusions: By this protocol, Oxygen Consumption Rate (OCR) measurements of fresh and cryopreserved placental specimens are comparable whereas snap frozen procedure impairs mitochondrial activity. [ABSTRACT FROM AUTHOR]

Background: Arbuscular mycorrhizal fungi (AMF) can regulate metal(loid) tolerance in plants and their capacity for phytoremediation. These effects can vary depending on the host plant and the AMF species. The impact of different AMF species on the ability of safflower (Carthamus tinctorius L.) for arsenic (As) phytoremediation is still largely unknown. Therefore, this study aimed to assess the effect of two AMF species, Rhizophagus irregularis, and Funneliformis mosseae, on the tolerance and accumulation of As in safflower in soils spiked with varying arsenate concentrations (0, 25, 50, and 100 mg kg−1). Results: The results indicated that both AMF species established effective symbiotic relationships with safflower. However, plants inoculated with R. irregularis exhibited higher mycorrhizal dependency and root colonization, especially under 100 mg kg−1 As. Both AMF species significantly improved plant growth parameters, chlorophyll content, and phosphorus (P) nutrition, which resulted in increased P/As ratio and enhanced tolerance index in safflower plants. In addition, AMF inoculation reduced As-induced lipid peroxidation by enhancing catalase and peroxidase activity in leaves and roots. While the mycorrhizal symbiosis didn't affect As availability in soils, it significantly reduced shoot As concentration and the translocation factor under all As levels. Furthermore, mycorrhizal inoculation, especially with R. irregularis, increased As concentration and modified-bioconcentration factor in the roots and enhanced total As uptake per plant. Conclusions: Based on the results and multivariate analyses, both AMF species, particularly R. irregularis, enhanced safflower's As tolerance by retaining As in roots, improving phosphorus nutrition, and increasing antioxidant enzyme activity, showcasing their potential to enhance phytostabilization in safflower plants. [ABSTRACT FROM AUTHOR]

Background: Viola diffusa is used in the formulation of various Traditional Chinese Medicines (TCMs), including antiviral, antimicrobial, antitussive, and anti-inflammatory drugs, due to its richness in flavonoids and triterpenoids. The biosynthesis of these compounds is largely mediated by cytochrome P450 enzymes, which are primarily located in the membranes of mitochondria and the endoplasmic reticulum. Results: This study presents the complete assembly of the mitogenome and plastome of Viola diffusa. The circular mitogenome spans 474,721 bp with a GC content of 44.17% and encodes 36 unique protein-coding genes, 21 tRNA, and 3 rRNA. Except for the RSCU values of 1 observed for the start codon (AUG) and tryptophan (UGG), the mitochondrial protein-coding genes exhibited a codon usage bias, with most estimates deviating from 1, similar to patterns observed in closely related species. Analysis of repetitive sequences in the mitogenome demonstrated potential homologous recombination mediated by these repeats. Sequence transfer analysis revealed 24 homologous sequences shared between the mitogenome and plastome, including nine full-length genes. Collinearity was observed among Viola diffusa species within the other members of Malpighiales order, indicated by the presence of homologous fragments. The length and arrangement of collinear blocks varied, and the mitogenome exhibited a high frequency of gene rearrangement. Conclusions: We present the first complete assembly of the mitogenome and plastome of Viola diffusa, highlighting its implications for pharmacological, evolutionary, and taxonomic studies. Our research underscores the multifaceted importance of comprehensive mitogenome analysis. [ABSTRACT FROM AUTHOR]

Background: South China carp (Cyprinus carpio rubrofuscus), which is an economically important species, is traditionally cocultured with rice. Our previous study indicated that approximately 10–30% of these fish experienced growth arrest, severely impacting production. However, the molecular mechanism underlying growth inhibition in south China carp is currently unknown. Results: In this study, we compared the transcriptomes of the livers, muscles and intestines of carp in the fast-growing and slow-growing groups. We identified 2182, 2355 and 916 differentially expressed genes (DEGs), respectively. In the slow-growing group, the oxidative phosphorylation pathway was significantly upregulated in the liver. Transmission electron microscopy (TEM) confirmed mitochondrial damage in the liver, which was characterized by broken cristae and heterogeneous matrix. Additionally, analysis of antioxidant enzyme and transaminase activity also revealed that the livers in slow-growing individuals were unhealthy. In muscle tissue, the mitophagy and autophagy pathways were significantly dysregulated. Consequently, manifestations of mitochondrial damage and sparse myofilaments were clearly observed in slow-growing south China carp via TEM. Furthermore, pathways that regulate cell proliferation and migration, including the ECM receptor and focal adhesion, were significantly enriched in the intestine. Morphological examination revealed that the villus height and muscular layer height in the slow-growing group were significantly shorter than those in the fast-growing group, suggesting decreased intestinal cell motility. Overall, our study elucidated mitochondrial damage in the liver and muscle and detected morphological changes in intestinal villi. Conclusions: In summary, our results help elucidate the genetic architecture related to growth arrest in south China carp and provide a basis for further research on the growth of teleosts. [ABSTRACT FROM AUTHOR]

Background: Dorper sheep are celebrated for their fast maturation and superior meat quality, with some shedding their wool each spring. Wool shedding occurs naturally due to the hair follicle (HF) cycle, but its regulatory mechanisms remain unclear and need further investigation. Results: In this study, shedding and non-shedding sheep were selected from the same Dorper flock. Skin samples were collected in September of the first year and January and March of the following years. RNA sequencing was performed on these samples. Principal component analysis (PCA) was used to assess the results. A total of 2536 differentially expressed genes (DEGs) were identified. Using a clustering heatmap and fuzzy clustering analysis three distinct gene expression patterns were identified: A pattern (high expression in anagen), T1 pattern, and T2 pattern (high expression in telogen). For each pattern, differentially expressed genes (DEGs) were analyzed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Combining this with pathway expression analysis, six A-pattern and fourteen T-pattern pathways linked to telogen-anagen transition in the HF cycle were identified. Networks of key pathways were then constructed. Additionally, key genes were identified in the telogen-anagen transition, including one A-pattern gene and seven T-pattern (T1, 1; T2, 6) genes, using the Maximal Clique Centrality (MCC) tool in Cytoscape. Predicted transcription factors (TFs) involved in key pathways, such as LEF and STAT5B, were identified. Finally, RNA-seq results were confirmed by RT-qPCR. Conclusion: This study highlights critical genes and pathways in the telogen-anagen transition, and transcriptome sequencing along with bioinformatics analysis provides new insights into the regulatory mechanisms of the HF cycle and development. [ABSTRACT FROM AUTHOR]

Background: Hypoxia negatively affects the behavior, growth, reproduction and survival of fish, causing serious economic losses to aquaculture. Rainbow trout (Oncorhynchus mykiss), an important economic fish worldwide, belongs to a hypoxia-sensitive fish species, however, little is known about the regulatory mechanism of microRNAs (miRNAs) under hypoxia stress. Results: Rainbow trout were subjected to hypoxia stress for 3 h (H3h_L), 12 h (H12h_L), 24 h (H24h_L) and 3 h reoxygenation (R3h_L) to systemically evaluate the changes of miRNA expression profiles in liver, and functions of sha-miR-92a_L + 2R + 4 were investigated. We found 17, 144, 57 and 55 differentially expressed (DE) miRNAs in the H3h_L vs. control (N_L), H12h_L vs. N_L, H24h_L vs. N_L and R3h_L vs. N_L comparisons, respectively. Enrichment analysis revealed that the targets of DE miRNAs were significantly enriched in HIF signaling pathway, VEGF signaling pathway, FoxO signaling pathway and glycolysis/gluconeogenesis. Through miRNA-mRNA interaction and weighted gene co-expression network analysis (WGCNA), five key DE miRNAs (sha-miR-92a_L + 2R + 4, ssa-miR-128-3p, ssa-miR-101b-3p_R + 1, ola-miR-199a-5p_R + 2 and tni-miR-199_1ss18CG) were identified, which can target at least two hypoxia-responsive genes, such as vegfaa, ho, glut1a and junb. Functional analysis found that sha-miR-92a_L + 2R + 4 directly regulated vegfaa expression by targeting its 3′-UTR, overexpression of sha-miR-92a_L + 2R + 4 significantly decreased vegfaa expression in rainbow trout liver cells, while opposite results were obtained after transfection of sha-miR-92a_L + 2R + 4 inhibitor. Furthermore, overexpressed sha-miR-92a_L + 2R + 4 promoted rainbow trout liver cell proliferation and inhibited apoptosis. Conclusion: This study deepens our understanding of the crucial roles of miRNAs under hypoxia stress in rainbow trout. These results can contribute to devise strategies for improving rainbow trout survival rate and aquaculture production during hypoxia stress and help speeding up the selective breeding of hypoxia-tolerant rainbow trout. [ABSTRACT FROM AUTHOR]

Background: Liver metastasis poses a significant barrier to effective immunotherapy in patients with colorectal cancer. Cryoablation has emerged as a vital supplementary therapeutic approach for these patients. However, its impact on the tumor microenvironment following the ablation of liver metastases remains unclear. Methods: We acquired multi-omics time-series data at 1 day, 5 days, and 14 days post-cryoablation, based on tumor and peripheral blood samples from clinical patients, cell co-culture models, and a liver metastases mouse model built on the MC38 cell line in C57BL/6 J mice. This dataset included single-cell transcriptomic sequencing, bulk tissue transcriptomic sequencing, 4D-Label-Free proteomics, flow cytometry data, western blot data, and histological immunofluorescence staining of pathological specimens. Results: We found that a neutrophil-related inflammatory state persisted for at least 14 days post-cryoablation. During this period, neutrophils underwent phenotypic changes, shifting from the N1 to the N2 type. Cryoablation also caused a significant increase in intracellular Ca2+ concentration in neutrophils, which triggered the formation of PAD4-dependent neutrophil extracellular traps (NETs), further promoting immune evasion. Moreover, animal studies demonstrated that depleting or inhibiting the CXCL2-CXCR2 signaling axis within neutrophils, or degrading NETs, could effectively restore the host's anti-tumor immune response. Conclusions: These findings underscore the critical role of neutrophils and their NETs in immune escape following cryoablation. Targeting the CXCL2-CXCR2-Ca2+-PAD4 axis could enhance the therapeutic response to PD-1 antibodies, providing a potential strategy to improve treatment outcomes for colorectal cancer with liver metastases. [ABSTRACT FROM AUTHOR]

Background: Granulosa cell (GC) apoptosis, ferroptosis, and other programmed cell death processes are markers of follicular aging. Quercetin has been shown to reduce ferroptosis, however, its effects on ferroptosis in poultry remains unexplored. Our preliminary study identified ferroptosis in aging ovaries. Therefore, in the present study, 540-day-old Mountain Plum-blossom chickens were fed with quercetin supplementation at varying doses (0.2, 0.4, and 0.6 g/kg), and examined its molecular effects on GC ferroptosis using an in vitro Erastin-induced model. Results: The results showed that quercetin supplementation significantly increased egg production, which confirmed its potential to alleviate ferroptosis in chicken ovarian tissue. The in vitro experiment revealed that quercetin and Fer-1 (positive control) mitigated Erastin-induced ferroptosis in GCs. Further, transcriptome analysis revealed that quercetin modulated key genes such as acyl-CoA synthetase long-chain family member 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11), and transferrin receptor (TFRC), involved in ferroptosis regulation. The results further showed that quercetin also reduced Erastin-induced apoptosis and inflammation by modulating the expression of genes and proteins related to apoptosis and inflammatory factors (NF-κB, TNF-α, IL-6, and IL-10). Conclusion: Taken together, the results showed that quercetin improves egg production performance in chickens and mitigates ovarian ferroptosis in aging hens, and inhibits Erastin-induced ferroptosis, inflammation, and apoptosis in GCs. These findings revealed the protective role of quercetin in poultry ovarian tissue and its cellular mechanisms against detrimental factors in poultry production. [ABSTRACT FROM AUTHOR]

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Background: The circadian clock integrates external environmental changes into the internal physiology of organisms. Perturbed circadian clocks due to misaligned light cycles increase the risk of diseases, including metabolic disorders. However, the effects of sex differences in this context remain unclear. Methods: Circadian misalignment was induced by a chronic jet lag (CJL) shift schedule (light-on time advanced by 6 h every 2 days) in C57BL/6N male and female mice. Core body temperature and activity rhythms were recorded using a nano tag, and the gene expression rhythms of clock and clock-controlled genes in the liver and adrenal glands were analyzed using qPCR. Glucose metabolism and insulin response were evaluated using glucose tolerance, insulin sensitivity, and glucose response assays. Castration and testosterone replacement were performed to assess the fundamental role of testosterone in male phenotypes under CJL. Results: Under CJL treatment, male mice exhibited increased weight gain, whereas females exhibited decreased weight gain compared to that of the respective controls. CJL treatment induced a lower robustness of circadian rhythms in core body temperature and a weaker rhythm of clock gene expression in the liver and adrenal glands in females, but not in males. Only male mice exhibited glucose intolerance under CJL conditions, without the development of insulin resistance. Castrated mice without testosterone exhibited decreased weight gain and reduced robustness of body temperature rhythm, as observed in intact females. Testosterone replacement in castrated mice recovered the CJL-induced weight gain, robustness of temperature rhythm, and glucose intolerance observed in intact males. Conclusions: Significant sex-based differences were observed in circadian clock organization and metabolism under CJL. Testosterone plays a crucial role in maintaining the circadian clock and regulating CJL metabolism in males. Plain English Summary: This study investigated the effects of chronic jet lag (CJL) on male and female mice, revealing significant differences in their responses to frequent light-dark shifts that induce circadian disruptions. Male mice exhibited increased body weight gain under CJL conditions, while in female mice, it was decreased. CJL treatment weakened the circadian rhythms of body temperature and clock gene expression in peripheral organs in females, but not in males. Only male mice developed glucose intolerance without developing insulin resistance. When male mice were castrated, they experienced reduced weight gain and changes in their circadian rhythms similar to that of females, but replacing testosterone restored their weight gain and glucose intolerance. Overall, this research highlights significant differences between the sexes in regard to how CJL impacts the circadian clock and metabolism, emphasizing the important role of testosterone in regulasting these processes in males. Highlights: Chronic jet lag increased weight gain in male mice and decreased it in female mice. Female mice exhibited vulnerable circadian rhythms following chronic jet lag. Male mice exhibited glucose intolerance under chronic jet lag. Testosterone is crucial for maintaining circadian rhythm and metabolism in males. [ABSTRACT FROM AUTHOR]

In recent years, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have emerged as critical regulators in plant biology, governing complex gene regulatory networks. In the context of disease resistance in Hevea brasiliensis, the rubber tree, significant progress has been made in understanding its response to anthracnose disease, a serious threat posed by fungal pathogens impacting global rubber tree cultivation and latex quality. While advances have been achieved in unraveling the genetic and molecular foundations underlying anthracnose resistance, gaps persist in comprehending the regulatory roles of lncRNAs and miRNAs under such stress conditions. The specific contributions of these non-coding RNAs in orchestrating molecular responses against anthracnose in H. brasiliensis remain unclear, necessitating further exploration to uncover strategies that increase disease resistance. Here, we integrate lncRNA sequencing, miRNA sequencing, and degradome sequencing to decipher the regulatory landscape of lncRNAs and miRNAs in H. brasiliensis under anthracnose stress. We investigated the genomic and regulatory profiles of differentially expressed lncRNAs (DE-lncRNAs) and constructed a competitive endogenous RNA (ceRNA) regulatory network in response to pathogenic infection. Additionally, we elucidated the functional roles of HblncRNA29219 and its antisense hbr-miR482a, as well as the miR390-TAS3-ARF pathway, in enhancing anthracnose resistance. These findings provide valuable insights into plant-microbe interactions and hold promising implications for advancing agricultural crop protection strategies. This comprehensive analysis sheds light on non-coding RNA-mediated regulatory mechanisms in H. brasiliensis under pathogen stress, establishing a foundation for innovative approaches aimed at enhancing crop resilience and sustainability in agriculture. [ABSTRACT FROM AUTHOR]

Background: Osteoarthritis (OA) poses a significant risk to the mobility of patients. Carbonic anhydrase 12 (CA12) can boost apoptosis and inflammation in several cancers, but its role in OA is unknown. Methods: Differentially expressed genes in OA were analyzed using the GEO database (GSE169077). RT-qPCR and western blot estimated relative mRNA and protein levels of CA12. Cell viability and apoptosis were estimated by cell counting and flow cytometry assays. Oxidative stress (OxS) was determined by detecting with ROS and MDA levels, as well as CAT and SOD activities. Cytokine levels of IL-6 and TNF-α were detected by ELISA. Parameters associated with apoptosis and extracellular matrix (ECM) were detected by western blot. The m6A modification profile was determined by methylated RNA immunoprecipitation assays. Results: Relative CA12 and wilms' tumor 1-associating protein (WTAP) mRNA and protein levels were overexpressed in OA articular cartilages and IL-1β-challenged chondrocytes CHON-001. CA12 silencing impaired IL-1β-induced cell apoptosis, inflammation, OxS, and ECM degradation in chondrocytes. Yet, CA12 overexpression exerted an opposing function. WTAP reinforced the stability of CA12 mRNA depending on the m6A modification. Furthermore, WTAP knockdown weakened cell apoptosis, inflammation, OxS, and ECM degradation in chondrocytes induced by IL-1β, but these changes were impaired after CA12 overexpression. In addition, WTAP knockdown mitigates cartilage degeneration in DMM-induced mouse models. Conclusion: IL-1β-induced WTAP enhances CA12 mRNA stability depending on m6A modification, thus promoting chondrocyte apoptosis, inflammatory response, OxS, and ECM degradation, providing evidence to support the possibility of WTAP and CA12 as potential targets for OA treatment. [ABSTRACT FROM AUTHOR]

Background: The eradication of Plasmodium vivax malaria is complicated due to the presence of hypnozoites, the hidden dormant form of the parasite that is present in the liver. Currently available drug regimens are effective at killing hypnozoites but cause side effects and are difficult to administer. Studies testing drugs for liver-stage malaria remain rare and mainly rely on the use of cancerous or immortalized hepatic cells and primary hepatocytes. Methods: Organoids were used as platform to model liver-stage vivax malaria. Hepatic endoderm cells, endothelial progenitor cells and mesenchymal cells were generated from human induced pluripotent stem cells and self-assembled into liver organoids on top of Matrigel layer. Liver characteristic and maturity were examined through genes and proteins expression of liver markers, and liver functional tests before infected with Plasmodium vivax sporozoites. The infection was then verified by the detection of parasitophorous vacuole membrane proteins, Upregulated in Infectious Sporozoite 4 (UIS4), and blood-stage infection following co-culture with human reticulocytes. Results: Generated liver organoids showed upregulation of liver specific transcripts including hepatic nuclear factor 4A (HNF4A), alpha-fetoprotein (AFP), and albumin (ALB) which also confirmed by the protein expression. Furthermore, those organoids resembled mature hepatocytes in terms of albumin secretion, fat and glycogen storage and cytochrome activity. Following invasion of P. vivax sporozoites, PvUIS4 was detected and the hepatic merozoites could develop into ring-stage and early trophozoites in human reticulocytes. Moreover, differential expression patterns of genes involved in lipid and cholesterol synthesis were also detected. Conclusions: Stem cell-derived liver organoids resemble mature liver cells in terms of liver functions and are susceptible to infection with P. vivax sporozoites, paving the way for studies on the mechanism of hypnozoite formation and testing of possible hypnozoitocidal drugs. [ABSTRACT FROM AUTHOR]