Rosavin: Research Advances in Extraction and Synthesis, Pharmacological Activities and Therapeutic Effects on Diseases of the Characteristic Active Ingredients of Rhodiola rosea L.

Rhodiola rosea L. (RRL) is a popular plant in traditional medicine, and Rosavin, a characteristic ingredient of RRL, is considered one of the most important active ingredients in it. In recent years, with deepening research on its pharmacological actions, the clinical application value and demand for Rosavin have been steadily increasing. Various routes for the extraction and all-chemical or biological synthesis of Rosavin have been gradually developed for the large-scale production and broad application of Rosavin. Pharmacological studies have demonstrated that Rosavin has a variety of biological activities, including antioxidant, lipid-lowering, analgesic, antiradiation, antitumor and immunomodulation effects. Rosavin showed significant therapeutic effects on a range of chronic diseases, including neurological, digestive, respiratory and bone-related disorders during in vitro and vivo experiments, demonstrating the great potential of Rosavin as a therapeutic drug for diseases. This paper gives a comprehensive and insightful overview of Rosavin, focusing on its extraction and synthesis, pharmacological activities, progress in disease-treatment research and formulation studies, providing a reference for the production and preparation, further clinical research and applications of Rosavin in the future.

A close look at current γδ T-cell immunotherapy.

Owing to their antitumor and major histocompatibility complex (MHC)-independent capacities, γδ T cells have gained popularity in adoptive T-cell immunotherapy in recent years. However, many unknowns still exist regarding γδ T cells, and few clinical data have been collected. Therefore, this review aims to describe all the main features of the applications of γδ T cells and provide a systematic view of current γδ T-cell immunotherapy. Specifically, this review will focus on how γδ T cells performed in treating cancers in clinics, on the γδ T-cell clinical trials that have been conducted to date, and the role of γδ T cells in the pharmaceutical industry.

Magnetically Actuated Cell-Robot System: Precise Control, Manipulation, and Multimode Conversion.

Border-nearing microrobots with self-propelling and navigating capabilities have promising applications in micromanipulation and bioengineering, because they can stimulate the surrounding fluid flow for object transportation. However, ensuring the biosafety of microrobots is a concurrent challenge in bioengineering applications. Here, macrophage template-based microrobots (cell robots) that can be controlled individually or in chain-like swarms are proposed, which can transport various objects. The cell robots are constructed using the phagocytic ability of macrophages to load nanomagnetic particles while maintaining their viability. The robots exhibit high position control accuracy and generate a flow field that can be used to transport microspheres and sperm when exposed to an external magnetic field near a wall. The cell robots can also form chain-like swarms to transport a large object (more than 100 times the volume). This new insight into the manipulation of macrophage-based cell robots provides a new concept by converting other biological cells into microrobots for micromanipulation in biomedical applications.

Precise Control of Customized Macrophage Cell Robot for Targeted Therapy of Solid Tumors with Minimal Invasion.

Injecting micro/nanorobots into the body to kill tumors is one of the ultimate ambitions for medical nanotechnology. However, injecting current micro/nanorobots based on 3D-printed biocompatible materials directly into blood vessels for targeted therapy is often difficult, and mistakes in targeting can cause serious side effects, such as blood clots, oxidative stress, or inflammation. The natural affinity of macrophages to tumors, and their natural phagocytosis and ability to invade tumors, make them outstanding drug delivery vehicles for targeted tumor therapy. Hence, a magnetically controlled cell robot (MCR) based on a macrophage drug carrier is proposed. Here, living macrophages are converted into MCRs through endocytosis of specially-designed magnetic nanoparticles loaded with doxorubicin and indocyanine green. Following this, the MCRs can be transported to tumors through the blood vessels using external magnetic fields, and penetrate the blood vessels into the interior of the tumor due to their deformability. With the MCR's cascaded drug release, targeted killing of tumors in mice is demonstrated, with minimal effects on the normal surrounding tissue. The ability to impart precise drug doses onto natural cells, such as macrophages, and load various functional components into the MCRs, offers an efficient method for precise targeted therapy.

Alternate-Layered MXene Composite Film-Based Triboelectric Nanogenerator with Enhanced Electrical Performance.

The output power of the triboelectric nanogenerator (TENG) strongly depends on the performance of triboelectric materials, especially microstructures and functional groups of them. In this work, aiming at the excellent triboelectric ability, alternate-layered MXene composite films-based TENG with abundant fluorine groups(-F) through layer-by-layer stacking are designed and fabricated. Benefitting from the uniform intrinsic microstructure and increased dielectric constant, when the amount of the Nb2CTx nanosheets increases to 15 wt%, the TENG based on Nb2CTx/Ti3C2Tx composite nanosheets films achieves the maximum output. The short-circuit current density of 8.06 µA/cm2 and voltage of 34.63 V are 8.4 times and 3.5 times over that of pure Ti3C2Tx films, and 3.3 times and 4.3 times over that of commercial poly(tetrafluoroethylene) (PTFE) films, respectively. Furthermore, the fabricated TENG could be attached to human body to harvest energy from human motions, such as typing, texting, and hand clapping. The results demonstrate that the alternate-layered MXene composite nanosheet films through layer-by-layer stacking possess remarkably triboelectric performance, which broaden the choice of negative triboelectric materials and supply a new choice for high output TENG.

The Basic Properties of Gold Nanoparticles and their Applications in Tumor Diagnosis and Treatment.

Gold nanoparticles (AuNPs) have been widely studied and applied in the field of tumor diagnosis and treatment because of their special fundamental properties. In order to make AuNPs more suitable for tumor diagnosis and treatment, their natural properties and the interrelationships between these properties should be systematically and profoundly understood. The natural properties of AuNPs were discussed from two aspects: physical and chemical. Among the physical properties of AuNPs, localized surface plasmon resonance (LSPR), radioactivity and high X-ray absorption coefficient are widely used in the diagnosis and treatment of tumors. As an advantage over many other nanoparticles in chemicals, AuNPs can form stable chemical bonds with S-and N-containing groups. This allows AuNPs to attach to a wide variety of organic ligands or polymers with a specific function. These surface modifications endow AuNPs with outstanding biocompatibility, targeting and drug delivery capabilities. In this review, we systematically summarized the physicochemical properties of AuNPs and their intrinsic relationships. Then the latest research advancements and the developments of basic research and clinical trials using these properties are summarized. Further, the difficulties to be overcome and possible solutions in the process from basic laboratory research to clinical application are discussed. Finally, the possibility of applying the results to clinical trials was estimated. We hope to provide a reference for peer researchers to better utilize the excellent physicochemical properties of gold nanoparticles in oncotherapy.

Magnetically Driven Bionic Millirobots with a Low-Delay Automated Actuation System for Bioparticles Manipulation.

This paper presents a semi-automatic actuation system which can achieve bio-particles tracking, transportation, and high-precision motion control of robots in a microfluidic chip. This system is mainly applied in magnetically driven robots. An innovative manta ray-like robot was designed to increase stability of robots in a non-contaminated manipulation environment. A multilayer piezo actuator was applied to generate high-frequency vibration to decrease the friction between robots and the glass substrate. We also set up a user-friendly GUI (Graphical User Interface) and realized robot tracking and predetermined trajectory motion through excellent algorithms using Python and C++. In biotechnology, precise transportation of cells is used for the enucleation, microinjection, and investigation of the characteristics of a single cell. Being optimized, the parameters of the robot can effectively reach 10 µm in actuation precision and a maximum actuation speed of 200 mm/s.

On-chip rotational manipulation of microbeads and oocytes using acoustic microstreaming generated by oscillating asymmetrical microstructures.

The capability to precisely rotate cells and other micrometer-sized biological samples is invaluable in biomedicine, bioengineering, and biophysics. We propose herein a novel on-chip cell rotation method using acoustic microstreaming generated by oscillating asymmetrical microstructures. When the vibration is applied to a microchip with our custom-designed microstructures, two different modes of highly localized microvortices are generated that are utilized to precisely achieve in-plane and out-of-plane rotational manipulation of microbeads and oocytes. The rotation mechanism is studied and verified using numerical simulations. Experiments of the microbeads are conducted to evaluate the claimed functions and investigate the effects of various parameters, such as the frequency and the driving voltage on the acoustically induced flows. Accordingly, it is shown that the rotational speed and direction can be effectively tuned on demand in single-cell studies. Finally, the rotation of swine oocytes is involved as further applications. By observing the maturation stages of M2 after the exclusion of the first polar body of operated oocytes, the proposed method is proved to be noninvasive. Compared with the conventional approaches, our acoustofluidic cell rotation approach can be simple-to-fabricate and easy-to-operate, thereby allowing rotations irrespective of the physical properties of the specimen under investigation.

Cell Injection Millirobot Development and Evaluation in Microfluidic Chip.

We propose an innovative design of millirobot, which can achieve donor cell suction, delivery, and injection in a mammalian oocyte on a microfluidic chip. The millirobot body contains a hollow space that produces suction and ejection forces for the injection of cell nuclei using a nozzle at the tip of the robot. Specifically, a controller changes the hollow volume by balancing the magnetic and elastic forces of a membrane along with the motion of stages in the XY plane. A glass capillary attached to the tip of the robot contains a nozzle that is able to absorb and inject cell nuclei. The millirobot provides three degrees of freedom and generates micronewton forces. We demonstrate the effectiveness of the proposed millirobot through an experiment of the absorption and ejection of 20-µm particles from the nozzle using magnetic control in a microfluidic chip.

The transcription factor SOX30 is a key regulator of mouse spermiogenesis.

The postmeiotic development of male germ cells, also known as spermiogenesis, features the coordinated expression of a large number of spermatid-specific genes. However, only a limited number of key transcription factors have been identified and the underlying regulatory mechanisms remain largely unknown. Here, we report that SOX30, the most-divergent member of the Sry-related high-motility group box (SOX) family of transcription factors, is essential for mouse spermiogenesis. The SOX30 protein was predominantly expressed in spermatids, while its transcription was regulated by retinoic acid and by MYBL1 before and during meiosis. Sox30 knockout mice arrested spermiogenesis at step 3 round spermatids, which underwent apoptosis and abnormal chromocenter formation. We also determined that SOX30 regulated the expression of hundreds of spermatid-specific protein-coding and long non-coding RNA genes. SOX30 bound to the proximal promoter of its own gene and activated its transcription. These results reveal SOX30 as a novel key regulator of spermiogenesis that regulates its own transcription to enforce and activate this meiotic regulatory pathway.

Reprogramming p53-Deficient Germline Stem Cells Into Pluripotent State by Nanog.

Cultured mouse spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), revert back to pluripotent state either spontaneously or upon being modified genetically. However, the reprogramming efficiencies are low, and the underlying mechanism remains poorly understood. In the present study, we conducted transcriptomic analysis and found that many transcription factors and epigenetic modifiers were differentially expressed between GSCs and embryonic stem cells. We failed in reprogramming GSCs to pluripotent state using the Yamanaka 4 Factors, but succeeded when Nanog and Tet1 were included. More importantly, reprogramming was also achieved with Nanog alone in a p53-deficient GSC line with an efficiency of 0.02‰. These GSC-derived-induced pluripotent stem cells possessed in vitro and in vivo differentiation abilities despite the low rate of chimera formation, which might be caused by abnormal methylation in certain paternally imprinted genes. Together, these results show that GSCs can be reprogrammed to pluripotent state via multiple avenues and contribute to our understanding of the mechanisms of GSC reprogramming.

Gene knockout of Zmym3 in mice arrests spermatogenesis at meiotic metaphase with defects in spindle assembly checkpoint.

ZMYM3, a member of the MYM-type zinc finger protein family and a component of a LSD1-containing transcription repressor complex, is predominantly expressed in the mouse brain and testis. Here, we show that ZMYM3 in the mouse testis is expressed in somatic cells and germ cells until pachytene spermatocytes. Knockout (KO) of Zmym3 in mice using the CRISPR-Cas9 system resulted in adult male infertility. Spermatogenesis of the KO mice was arrested at the metaphase of the first meiotic division (MI). ZMYM3 co-immunoprecipitated with LSD1 in spermatogonial stem cells, but its KO did not change the levels of LSD1 or H3K4me1/2 or H3K9me2. However, Zmym3 KO resulted in elevated numbers of apoptotic germ cells and of MI spermatocytes that are positive for BUB3, which is a key player in spindle assembly checkpoint. Zmym3 KO also resulted in up-regulated expression of meiotic genes in spermatogonia. These results show that ZMYM3 has an essential role in metaphase to anaphase transition during mouse spermatogenesis by regulating the expression of diverse families of genes.

MicroRNA-202 maintains spermatogonial stem cells by inhibiting cell cycle regulators and RNA binding proteins.

miRNAs play important roles during mammalian spermatogenesis. However, the function of most miRNAs in spermatogenesis and the underlying mechanisms remain unknown. Here, we report that miR-202 is highly expressed in mouse spermatogonial stem cells (SSCs), and is oppositely regulated by Glial cell-Derived Neurotrophic Factor (GDNF) and retinoic acid (RA), two key factors for SSC self-renewal and differentiation. We used inducible CRISPR-Cas9 to knockout miR-202 in cultured SSCs, and found that the knockout SSCs initiated premature differentiation accompanied by reduced stem cell activity and increased mitosis and apoptosis. Target genes were identified with iTRAQ-based proteomic analysis and RNA sequencing, and are enriched with cell cycle regulators and RNA-binding proteins. Rbfox2 and Cpeb1 were found to be direct targets of miR-202 and Rbfox2 but not Cpeb1, is essential for the differentiation of SSCs into meiotic cells. Accordingly, an SSC fate-regulatory network composed of signaling molecules of GDNF and RA, miR-202 and diverse downstream effectors has been identified.

BMP4 Cooperates with Retinoic Acid to Induce the Expression of Differentiation Markers in Cultured Mouse Spermatogonia.

Spermatogenesis is sustained by the proliferation and differentiation of spermatogonial stem cells (SSCs). However, the molecules controlling these processes remain largely unknown. Here, we developed a simplified high concentration serum-containing system for the culture of mouse SSCs. Analysis of SSCs markers and transplantation results revealed that the cultured spermatogonia retained stem cell characteristics after long-term in vitro propagation. Using this culture system, the expression and function of bone morphogenetic protein 4 (BMP4) were explored. Immunostaining showed that BMP4 was predominantly expressed in germ cells and that its level increased as spermatogenesis progresses. BMP4 receptors BMPR1A and BMPRII were present in spermatogonia, spermatocytes, and round spermatids. Moreover, despite the mRNAs of these two genes being present in mouse Sertoli cells, only BMPRII was detected by using Western blotting assays. While exogenous BMP4 by itself did not induce the expression of Stra8 and c-Kit, two marker genes of differentiating spermatogonia, a significant cooperative effect of BMP4 and retinoic acid (RA) was observed. Moreover, pretreatment of cultured spermatogonia with the BMP4 antagonist Noggin could inhibit RA-induced expression of these two marker genes. In conclusion, BMP4 may exert autocrine effects and act cooperatively with RA to induce the differentiation of spermatogonia in vivo.

Retinoic Acid Is Sufficient for the In Vitro Induction of Mouse Spermatocytes.

Meiosis is the key step in gametogenesis. However, the mechanism of mammalian meiosis remains poorly understood due to the lack of an in vitro model. Here, we report that retinoic acid (RA) is sufficient for inducing leptotene/zygotene spermatocytes from cultured mouse spermatogonial stem cells. Multiple genes regulated by RA were identified by RNA sequencing. RA in combination with pup Sertoli cell co-culture resulted in a higher induction efficiency of 28%. Comparisons in the transcriptomic profiles of the induced spermatogenic cells and the isolated ones revealed the progressive induction of the germ cells. Using this model, we showed that Stra8, Agpat3, Fam57a, Wdr91, and Sox30 contributed to the proliferation and meiosis initiation differentially. In conclusion, we have efficiently generated spermatocytes using an RA/pup Sertoli cell-based in vitro model and provided proof-of-concept evidence for its application in identifying genes involved in mammalian meiosis.

Exposure to Brefeldin A promotes initiation of meiosis in murine female germ cells.

In mammals, ontogenesis starts from a fusion of spermatozoon and oocyte, which are produced by reductive nuclear division of a diploid germ cell in a specialised but complex biological process known as meiosis. However, little is known about the mechanism of meiotic initiation in germ cells, although many factors may be responsible for meiosis both in male and female gonads. In this study, 11.5 days post coitum (dpc) female fetal mouse genital ridges were cultured in vitro with exposure to Brefeldin A (BFA) for 6h, and the changes in meiosis were detected. Synaptonemal-complex analysis implied that BFA played a positive role in meiosis initiation and this hypothesis was confirmed by quantitative PCR of meiosis-specific genes: stimulated by retinoic acid gene 8 (Stra8) and deleted in a zoospermia-like (DAZL). At the same time, mRNA expression of retinoic acid synthetase (Raldh2) and retinoic acid (RA) receptors increased in female gonads with in vitro exposure to BFA. Transplanting genital ridges treated with BFA into the kidney capsule of immunodeficient mice demonstrated that the development capacity of female germ cells was normal, while formation of primordial follicles was seen to be a result of accelerated meiosis after exposure to BFA. In conclusion, the study indicated that BFA stimulated meiosis initiation partly by RA signalling and then promoted the development of follicles.

Notch pathway regulates female germ cell meiosis progression and early oogenesis events in fetal mouse.

A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.

Effects of diethylhexyl phthalate (DEHP) given neonatally on spermatogenesis of mice.

We previously demonstrated that the effects of diethylhexyl phthalate (DEHP) alter reproduction function on male mice. Immature male mice were treated daily with DEHP from postnatal day 7-21, 7-35, 7-49, in a dose-dependent manner. As results, both the quality and quantity of spermatozoa were decreased in 60-day-old mice. The results by RT-PCR analysis indicated that DDx3Y, Usp9Y, RBM, E1F1AY, EGF, FSHR and EGFR genes were down-regulated, and LHR, Cyp17a1 and Cyp19a1 were down-regulated in response to DEHP. These genes were selected based on their markedly increased or decreased expression levels. However, DEHP had no effect on the meiotic process and recombination levels in male mouse germ cells. Treatment with DEHP induced histopathological changes in the testes. Taken together, these results provide a new insight into the molecular mechanisms underlying the detrimental impacts of DEHP in humans and wildlife.

Exposure to bisphenol A results in a decline in mouse spermatogenesis.

Bisphenol A (BPA), a chemical used in many consumer products, interferes with the endocrine system of mammals, including humans. The aim of the present study was to investigate the effect of BPA on spermatogenesis and semen quality. The objective of this study was to assess the effects of BPA on mouse spermatogenesis. CD1 mice were used in all experiments. Mice were treated with different doses of BPA (0, 20 and 40 µg kg⁻¹ day⁻¹ from postnatal Day (PND) 3 to PND21, PND 35 or PND49. After 5 weeks BPA treatment, oestrogen receptor α expression was increased in mouse testis, whereas the meiotic progression of germ cells was slowed. Thus, both the quality and quantity of spermatozoa were decreased in 7-week-old mice. However, BPA had no effect on DNA methylation of imprinted genes such as Igf2, Igf2r, Peg3 and H19, in germ cells. In addition, exposure of male mice to BPA resulted in abnormal offspring that were smaller with a low-quality pelage when they were 35 days old. In conclusion, BPA hampers spermatogenesis and the subsequent development of offspring.

Recovery of functional oocytes from cultured premeiotic germ cells after kidney capsule transplantation.

The efficiency of in vitro culture systems for a premeiotic female germ cell is still low, mostly because of our incomplete understanding of the mechanisms controlling oogenesis and the obvious difficulties in reproducing the complex in vivo environment of such a process under in vitro conditions. Here we explored the possibility of recovering the developmental potential of mouse oocytes generated in vitro from premeiotic germ cells by transplantation under a kidney capsule of adult animals. To this aim, mouse embryonic ovaries of 12.5 days postcoitum cultured in vitro in a serum-free medium for 7 or 14 days, were transplanted beneath the kidney capsule of immunodeficient mice and analyzed after 21 (7+21 group) or 14 days (14+14 group). Cultured ovaries before transplantation showed delayed oocyte meiotic progression and follicle development. Interestingly, grafted ovaries of both groups, especially those of the 7+21 group, seemed able to restore the reproductive cycle of recipients. While the almost complete absence of primordial follicles was observed in grafted ovaries, oocytes from these ovaries showed transcript levels of genes associated to oocyte maturation similar to control. Moreover, the developmental stage of follicles and oocytes of the 7+21 group ovaries were comparable to that of 21 days post partum in vivo ovaries, whereas significant developmental delay were found in the 14+14 group ovaries. Nevertheless, oocytes retrieved from transplanted ovaries of both groups matured (around 80%) and were fertilized in vitro (around 20%-45%). Two-cell embryos from the fertilized oocytes developed to hatching blastocysts (about 50%) or gave rise to healthy live offspring (from 6% to 10%) when transplanted in a host mother. In conclusion, our results indicate that premeiotic female germ cells cultured in vitro up to primordial/primary follicle stages preserve their capability to complete oogenesis and can be fertilized and generate live pups after transplantation into a suitable in vivo environment.