The regulatory relationship between RagA and Nprl2 in Drosophila gut development / 生物工程学报

The gastrointestinal tract is the largest digestive organ and the largest immune organ and detoxification organ, which is vital to the health of the body. Drosophila is a classic model organism, and its gut is highly similar to mammalian gut in terms of cell composition and genetic regulation, therefore can be used as a good model for studying gut development. target of rapmaycin complex 1 (TORC1) is a key factor regulating cellular metabolism. Nprl2 inhibits TORC1 activity by reducing Rag GTPase activity. Previous studies have found that nprl2 mutated Drosophila showed aging-related phenotypes such as enlarged foregastric and reduced lifespan, which were caused by over-activation of TORC1. In order to explore the role of Rag GTPase in the developmental defects of the gut of nprl2 mutated Drosophila, we used genetic hybridization combined with immunofluorescence to study the intestinal morphology and intestinal cell composition of RagA knockdown and nprl2 mutated Drosophila. The results showed that RagA knockdown alone could induce intestinal thickening and forestomach enlargement, suggesting that RagA also plays an important role in intestinal development. Knockdown of RagA rescued the phenotype of intestinal thinning and decreased secretory cells in nprl2 mutants, suggesting that Nprl2 may regulate the differentiation and morphology of intestinal cells by acting on RagA. Knockdown of RagA did not rescue the enlarged forestomach phenotype in nprl2 mutants, suggesting that Nprl2 may regulate forestomach development and intestinal digestive function through a mechanism independent of Rag GTPase.

Role of the Voltage-Gated Proton Channel Hv1 in Nervous Systems / 神经科学通报·英文版

Hv1 is the only voltage-gated proton-selective channel in mammalian cells. It contains a conserved voltage-sensor domain, shared by a large class of voltage-gated ion channels, but lacks a pore domain. Its primary role is to extrude protons from the cytoplasm upon pH reduction and membrane depolarization. The best-known function of Hv1 is the regulation of cytosolic pH and the nicotinamide adenine dinucleotide phosphate oxidase-dependent production of reactive oxygen species. Accumulating evidence indicates that Hv1 is expressed in nervous systems, in addition to immune cells and others. Here, we summarize the molecular properties, distribution, and physiological functions of Hv1 in the peripheral and central nervous systems. We describe the recently discovered functions of Hv1 in various neurological diseases, including brain or spinal cord injury, ischemic stroke, demyelinating diseases, and pain. We also summarize the current advances in the discovery and application of Hv1-targeted small molecules in neurological diseases. Finally, we discuss the current limitations of our understanding of Hv1 and suggest future research directions.

Electroacupuncture Alleviates Functional Constipation in Mice by Activating Enteric Glial Cell Autophagy via PI3K/AKT/mTOR Signaling / 中国结合医学杂志

OBJECTIVE@#To investigate autophagy-related mechanisms of electroacupuncture (EA) action in improving gastrointestinal motility in mice with functional constipation (FC).@*METHODS@#According to a random number table, the Kunming mice were divided into the normal control, FC and EA groups in Experiment I. The autophagy inhibitor 3-methyladenine (3-MA) was used to observe whether it antagonized the effects of EA in Experiment II. An FC model was established by diphenoxylate gavage. Then the mice were treated with EA stimulation at Tianshu (ST 25) and Shangjuxu (ST 37) acupoints. The first black stool defecation time, the number, weight, and water content of 8-h feces, and intestinal transit rate were used to assess intestinal transit. Colonic tissues underwent histopathological assessment, and the expressions of autophagy markers microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 were detected by immunohistochemical staining. The expressions of phosphoinositide 3-kinases (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) signaling pathway members were investigated by Western blot and quantitative reverse transcription-polymerase chain reaction, respectively. The relationship between enteric glial cells (EGCs) and autophagy was observed by confocal immunofluorescence microscopy, localization analysis, and electron microscopy.@*RESULTS@#EA treatment shortened the first black stool defecation time, increased the number, weight, and water content of 8-h feces, and improved the intestinal transit rate in FC mice (P<0.01). In terms of a putative autophagy mechanism, EA treatment promoted the expressions of LC3 and Beclin-1 proteins in the colonic tissue of FC mice (P<0.05), with glial fibrillary acidic protein (GFAP) and LC3 significantly colocalized. Furthermore, EA promoted colonic autophagy in FC mice by inhibiting PI3K/AKT/mTOR signaling (P<0.05 or P<0.01). The positive effect of EA on intestinal motility in FC mice was blocked by 3-MA.@*CONCLUSION@#EA treatment can inhibit PI3K/AKT/mTOR signaling in the colonic tissues of FC mice, thereby promoting EGCs autophagy to improve intestinal motility.

Baicalin Ameliorates Corticosterone-Induced Depression by Promoting Neurodevelopment of Hippocampal via mTOR/GSK3β Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the role of hippocampal neurodevelopment in the antidepressant effect of baicalin.@*METHODS@#Forty male Institute of Cancer Research mice were divided into control, corticosterone (CORT, 40 mg/kg), CORT+baicalin-L (25 mg/kg), CORT+baicalin-H (50 mg/kg), and CORT+fluoxetine (10 mg/kg) groups according to a random number table. An animal model of depression was established by chronic CORT exposure. Behavioral tests were used to assess the reliability of depression model and the antidepressant effect of baicalin. In addition, Nissl staining and immunofluorescence were used to evaluate the effect of baicalin on hippocampal neurodevelopment in mice. The protein and mRNA expression levels of neurodevelopment-related factors were detected by Western blot analysis and real-time polymerase chain reaction, respectively.@*RESULTS@#Baicalin significantly ameliorated the depressive-like behavior of mice resulting from CORT exposure and promoted the development of dentate gyrus in hippocampus, thereby reversing the depressive-like pathological changes in hippocampal neurons caused by CORT neurotoxicity. Moreover, baicalin significantly decreased the protein and mRNA expression levels of glycogen synthase kinase 3β (GSK3β), and upregulated the expression levels of cell cycle protein D1, p-mammalian target of rapamycin (mTOR), doublecortin, and brain-derived neurotrophic factor (all P<0.01). There were no significant differences between baicalin and fluoxetine groups (P>0.05).@*CONCLUSION@#Baicalin can promote the development of hippocampal neurons via mTOR/GSK3β signaling pathway, thus protect mice against CORT-induced neurotoxicity and play an antidepressant role.

Protective Effect and Mechanism of Kuntai Capsule on Angiotensin II -Induced Hypertension in Ovariectomized Rats / 中国结合医学杂志

OBJECTIVE@#To explore the protective effect and mechanism of Kuntai (KT) Capsule on angiotensin II (Ang II)-induced hypertension in ovariectomized (OVX) rats.@*METHODS@#Fifty-four rats were randomly divided into 6 groups according to a random number table, 9 in each group: control, OVX sham+Ang II, OVX, OVX+Ang II, OVX+Ang II +E2, and OVX+Ang II +KT. OVX rats model was constructed by retroperitoneal bilateral ovariectomy. After 4 weeks of pretreatment with KT Capsule [0.8 g/(kg·d) and 17- β -estradiol (E2, 1.2 mg/(kg·d)] respectively, Ang II was injected into a micro-osmotic pump with a syringe to establish a hypertensive rat model. Blood pressure of rat tail artery was measured in a wake state of rats using a non-invasive sphygmomanometer. Blood pressure changes were compared between the intervention groups (OVX+Ang II +KT, OVX+Ang II +E2) and the negative control group (OVX+Ang II). Serum malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected respectively. The expressions of oxidative stress-related protein superoxide dismutase2 (SOD2) and anti-thioredoxin (TRX), autophagy marker protein [beclin1, light chain (LC) 3 II/I ratio and autophagy canonical pathway protein phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (AKT)-mammalian target of rapamycin (mTOR)] were evaluated by Western blotting.@*RESULTS@#Compared with the OVX+Ang II group, the systolic blood pressure of OVX+Ang II +KT group was significantly lowered (P<0.05) but not the diastolic blood pressure. Besides, SOD2 and TRX protein levels in mycardial tissues were significantly reduced in the OVX+Ang II +KT group compared with the OVX+Ang II group (P<0.05). Oxidative stress serum markers MDA and SOD were down- and up-regulated in the OVX+Ang II +KT group, respectively (P<0.05). Compared with OVX+Ang II group, the levels of cardiac proteins beclin-1 and LC3II/LC3 I in OVX+Ang II +KT group were also up-regulated (P<0.05), and the expression levels of p-PI3K, p-AKT and mTOR protein were down-regulated (P<0.05).@*CONCLUSION@#KT could protect blood pressure of Ang II-induced OVX rats by inhibiting oxidative stress and up-regulating protective autophagy.

Meranzin Hydrate Improves Depression-Like Behaviors and Hypomotility via Ghrelin and Neurocircuitry / 中国结合医学杂志

OBJECTIVE@#To investigate whether meranzin hydrate (MH) can alleviate depression-like behavior and hypomotility similar to Chaihu Shugan Powder (CSP), and further explore the potential common mechanisms.@*METHODS@#Totally 120 Spraque-Dawley rats were randomly divided into 5-8 groups including sham, vehicle, fluoxetine (20 mg/kg), mosapride (10 mg/kg), CSP (30 g/kg), MH (9.18 mg/kg), [D-Lys3]-GHRP-6 (Dlys, 0.5 mg/kg), and MH+Dlys groups by a random number table, 8 rats in each group. And 32 mice were randomly divided into wild-type, MH (18 mg/kg), growth hormone secretagogue receptor-knockout (GHSR-KO), and GHSR+MH groups, 8 mice in each group. The forced swimming test (FST), open field test (OFT), tail suspension test (TST), gastric emptying (GE) test, and intestinal transit (IT) test were used to assess antidepressant and prokinetic (AP) effects after drug single administration for 30 min with absorbable identification in rats and mice, respectively. The protein expression levels of brain-derived neurotrophic factor (BDNF) and phosphorylated mammalian target of rapamycin (p-mTOR) in the hippocampus of rats were evaluated by Western blot. The differences in functional brain changes were determined via 7.0 T functional magnetic resonance imaging-blood oxygen level-dependent (fMRI-BOLD).@*RESULTS@#MH treatment improved depression-like behavior (FST, OFT) and hypomotility (GE, IT) in the acute forced swimming (FS) rats (all P<0.05), and the effects are similar to the parent formula CSP. The ghrelin antagonist [D-Lys3]-GHRP-6 inhibited the effect of MH on FST and GE (P<0.05). Similarly, MH treatment also alleviated depression-like behavior (FST, TST) in the wild-type mice, however, no effects were found in the GHSR KO mice. Additionally, administration of MH significantly stimulated BDNF and p-mTOR protein expressions in the hippocampus (both P<0.01), which were also prevented by [D-Lys3]-GHRP-6 (P<0.01). Besides, 3 main BOLD foci following acute FS rats implicated activity in hippocampus-thalamus-basal ganglia (HTB) circuits. The [D-Lys3]-GHRP-6 synchronously inhibited BOLD HTB foci. As expected, prokinetic mosapride only had effects on the thalamus and basal ganglia, but not on the hippocampus. Within the HTB, the hippocampus is implicated in depression and FD.@*CONCLUSIONS@#MH accounts for part of AP effects of parent formula CSP in acute FS rats, mainly via ghrelin-related shared regulation coupled to BOLD signals in brain areas. This novel functionally connection of HTB following acute stress, treatment, and regulation highlights anti-depression unified theory.

Research, development and application of collagen: a review / 生物工程学报

Collagen, which widely exists in skin, bone, muscle and other tissues, is a major structural protein in mammalian extracellular matrix. It participates in cell proliferation, differentiation, migration and signal transmission, plays an important role in tissue support and repair and exerts a protective effect. Collagen is widely used in tissue engineering, clinical medicine, food industry, packaging materials, cosmetics and medical beauty due to its good biological characteristics. This paper reviews the biological characteristics of collagen and its application in bioengineering research and development in recent years. Finally, we prospect the future application of collagen as a biomimetic material.

Zebrafish ambra1b knockout reveals a novel role for Ambra1 in primordial germ cells survival, sex differentiation and reproduction

BACKGROUND: AMBRA1 is an intrinsically disordered protein, working as a scaffold molecule to coordinate, by protein-protein interaction, many cellular processes, including autophagy, mitophagy, apoptosis and cell cycle progression. The zebrafish genome contains two ambra1 paralogous genes (a and b), both involved in development and expressed at high levels in the gonads. Characterization of the zebrafish paralogous genes mutant lines generated by CRISPR/Cas9 approach showed that ambra1b knockout leads to an all-male population. RESULTS: We demonstrated that the silencing of the ambra1b gene determines a reduction of primordial germ cells (PGCs), a condition that, in the zebrafish, leads to the development of all-male progeny. PGC reduction was confirmed by knockdown experiments and rescued by injection of ambra1b and human AMBRA1 mRNAs, but not ambra1a mRNA. Moreover, PGC loss was not rescued by injection with human AMBRA1 mRNA mutated in the CUL4-DDB1 binding region, thus suggesting that interaction with this complex is involved in PGC protection from loss. Results from zebrafish embryos injected with murine Stat3 mRNA and stat3 morpholino suggest that Ambra1b could indirectly regulate this protein through CUL4-DDB1 interaction. According to this, Ambra1+/- mice showed a reduced Stat3 expression in the ovary together with a low number of antral follicles and an increase of atretic follicles, indicating a function of Ambra1 in the ovary of mammals as well. Moreover, in agreement with the high expression of these genes in the testis and ovary, we found significant impairment of the reproductive process and pathological alterations, including tumors, mainly limited to the gonads. CONCLUSIONS: By exploiting ambra1a and ambra1b knockout zebrafish lines, we prove the sub-functionalization between the two paralogous zebrafish genes and uncover a novel function of Ambra1 in the protection from excessive PGC loss, which seems to require binding with the CUL4-DDB1 complex. Both genes seem to play a role in the regulation of reproductive physiology.

Mammalian DMRTs: Structure, function and relationship with cancer / 生理学报

DMRT, a gene family related to sexual determination, encodes a large group of transcription factors (DMRTs) with the double-sex and mab-3 (DM) domain (except for DMRT8), which is able to bind to and regulate DNAs. Current studies have shown that the DMRT gene family plays a critical role in the development of sexual organs (such as gender differentiation, gonadal development, germ cell development, etc.) as well as extrasexual organs (such as musculocartilage development, nervous system development, etc.). Additionally, it has been suggested that DMRTs may be involved in the cancer development and progression (such as prostate cancer, breast cancer, lung cancer, etc.). This review summarizes the research progress about the mammalian DMRTs' structure, function and its critical role in cancer development, progression and therapy (mainly in human and mice), which suggests that DMRT gene could be a candidate gene in the study of tumor formation and therapeutic strategy.

Roles of alternative splicing in infectious diseases: from hosts, pathogens to their interactions / 中华医学杂志(英文版)

Alternative splicing (AS) is an evolutionarily conserved mechanism that removes introns and ligates exons to generate mature messenger RNAs (mRNAs), extremely improving the richness of transcriptome and proteome. Both mammal hosts and pathogens require AS to maintain their life activities, and inherent physiological heterogeneity between mammals and pathogens makes them adopt different ways to perform AS. Mammals and fungi conduct a two-step transesterification reaction by spliceosomes to splice each individual mRNA (named cis -splicing). Parasites also use spliceosomes to splice, but this splicing can occur among different mRNAs (named trans -splicing). Bacteria and viruses directly hijack the host's splicing machinery to accomplish this process. Infection-related changes are reflected in the spliceosome behaviors and the characteristics of various splicing regulators (abundance, modification, distribution, movement speed, and conformation), which further radiate to alterations in the global splicing profiles. Genes with splicing changes are enriched in immune-, growth-, or metabolism-related pathways, highlighting approaches through which hosts crosstalk with pathogens. Based on these infection-specific regulators or AS events, several targeted agents have been developed to fight against pathogens. Here, we summarized recent findings in the field of infection-related splicing, including splicing mechanisms of pathogens and hosts, splicing regulation and aberrant AS events, as well as emerging targeted drugs. We aimed to systemically decode host-pathogen interactions from a perspective of splicing. We further discussed the current strategies of drug development, detection methods, analysis algorithms, and database construction, facilitating the annotation of infection-related splicing and the integration of AS with disease phenotype.

In vitro and in vivo anticancer potential and molecular targets of the new colchicine analog IIIM-067 / 中西医结合学报

OBJECTIVE@#The current study evaluated various new colchicine analogs for their anticancer activity and to study the primary mechanism of apoptosis and in vivo antitumor activity of the analogs with selective anticancer properties and minimal toxicity to normal cells.@*METHODS@#Sulforhodamine B (SRB) assay was used to screen various colchicine analogs for their in vitro cytotoxicity. The effect of N-[(7S)-1,2,3-trimethoxy-9-oxo-10-(pyrrolidine-1-yl)5,6,7,9-tetrahydrobenzo[a] heptalene-7-yl] acetamide (IIIM-067) on clonogenicity, apoptotic induction, and invasiveness of A549 cells was determined using a clonogenic assay, scratch assay, and staining with 4',6-diamidino-2-phenylindole (DAPI) and annexin V/propidium iodide. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels were observed using fluorescence microscopy. Western blot analysis was used to quantify expression of proteins involved in apoptosis, cell cycle, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling. Pharmacokinetic and in vivo efficacy studies against Ehrlich ascites carcinoma (EAC) and Ehrlich solid tumor models were conducted using Swiss albino mice.@*RESULTS@#IIIM-067 showed potent cytotoxicity and better selectivity than all other colchicine analogs screened in this study. The selective activity of IIIM-067 toward A549 cells was higher among other cancer cell lines, with a selectivity index (SI) value of 2.28. IIIM-067 demonstrated concentration- and time-dependent cytotoxicity against A549 cells with half-maximal inhibitory concentration values of 0.207, 0.150 and 0.106 μmol/L at 24, 48 and 72 h, respectively. It also had reduced toxicity to normal cells (SI > 1) than the parent compound colchicine (SI = 1). IIIM-067 reduced the clonogenic ability of A549 cells in a dose-dependent manner. IIIM-067 enhanced ROS production from 24.6% at 0.05 μmol/L to 82.1% at 0.4 μmol/L and substantially decreased the MMP (100% in control to 5.6% at 0.4 μmol/L). The annexin V-FITC assay demonstrated 78% apoptosis at 0.4 μmol/L. IIIM-067 significantly (P < 0.5) induced the expression of various intrinsic apoptotic pathway proteins, and it differentially regulated the PI3K/AKT/mTOR signaling pathway. Furthermore, IIIM-067 exhibited remarkable in vivo anticancer activity against the murine EAC model, with tumor growth inhibition (TGI) of 67.0% at a dose of 6 mg/kg (i.p.) and a reduced mortality compared to colchicine. IIIM-067 also effectively inhibited the tumor growth in the murine solid tumor model with TGI rates of 48.10%, 55.68% and 44.00% at doses of 5 mg/kg (i.p.), 6 mg/kg (i.p.) and 7 mg/kg (p.o.), respectively.@*CONCLUSION@#IIIM-067 exhibited significant anticancer activity with reduced toxicity both in vitro and in vivo and is a promising anticancer candidate. However, further studies are required in clinical settings to fully understand its potential.

Tinker, tailor, soldier, cell: the role of C-type lectins in the defense and promotion of disease

C-type lectins (CTLs) represent a large family of soluble and membrane-bound proteins which bind calcium dependently via carbohydrate recognition domains (CRDs) to glycan residues presented on the surface of a variety of pathogens. The deconvolution of a cell's glycan code by CTLs underpins several important physiological processes in mammals such as pathogen neutralization and opsonization, leukocyte trafficking, and the inflammatory response. However, as our knowledge of CTLs has developed it has become apparent that the role of this innate immune family of proteins can be double-edged, where some pathogens have developed approaches to subvert and exploit CTL interactions to promote infection and sustain the pathological state. Equally, CTL interactions with host glycoproteins can contribute to inflammatory diseases such as arthritis and cancer whereby, in certain contexts, they exacerbate inflammation and drive malignant progression. This review discusses the 'dual agent' roles of some of the major mammalian CTLs in both resolving and promoting infection, inflammation and inflammatory disease and highlights opportunities and emerging approaches for their therapeutic modulation.

Roles of NG2 Glia in Cerebral Small Vessel Disease / 神经科学通报·英文版

Cerebral small vessel disease (CSVD) is one of the most prevalent pathologic processes affecting 5% of people over 50 years of age and contributing to 45% of dementia cases. Increasing evidence has demonstrated the pathological roles of chronic hypoperfusion, impaired cerebral vascular reactivity, and leakage of the blood-brain barrier in CSVD. However, the pathogenesis of CSVD remains elusive thus far, and no radical treatment has been developed. NG2 glia, also known as oligodendrocyte precursor cells, are the fourth type of glial cell in addition to astrocytes, microglia, and oligodendrocytes in the mammalian central nervous system. Many novel functions for NG2 glia in physiological and pathological states have recently been revealed. In this review, we discuss the role of NG2 glia in CSVD and the underlying mechanisms.

Microorganisms capable of degrading neonicotinoids and their metabolic pathways: a review / 生物工程学报

Neonicotinoid compounds are usually considered harmless and eco-friendly in terms of their targeted toxicity compared to that of pyrethroids and phosphorus-containing pesticides. However, overuse of neonicotinoid insecticides resulted in the accumulation of its residuals or intermediates in soil and water, which consequently affected beneficial insects as well as mammals, yielding pollution and secondary risks. This review summarized the recent advances in neonicotinoid degrading microorganisms and their metabolic diversity, with the aim to address the urgent need for degrading these insecticides. These advances may facilitate the development of controllable and reliable technologies for efficiently transforming neonicotinoid insecticides into value-added products by synthetic biology and metagenomics.

Expression and role of the TRPC family in TGF-β1-induced calcium influx in podocytes / 生理学报

The TRPC family consists of multiple important cationic channels in mammals that participate in a variety of physiological and pathological processes. Our previous studies have shown that transforming growth factor-β1 (TGF-β1) increases the expression of TRPC6 in podocytes, but the roles of other members of the TRPC family in podocytes require further investigation. In this study, we investigated the effect of TGF-β1 on the expression of the TRPC family and the role of the TRPC family in the changes of the intracellular Ca2+ concentration ([Ca2+]i) in podocytes induced by TGF-β1. The model of podocyte injury was established by treatment with TGF-β1 in immortalized glomerular podocytes (MPC5) in vitro. qRT-PCR and Western blot were used to detect the effect of TGF-β1 on the mRNA and protein expression of each TRPC family member. After the expression of each TRPC family member was knocked down by a siRNA-based approach and blocked by SKF96365, respectively, free cytosolic Ca2+ was measured using the fluorescent Ca2+ indicator Fluo-3/AM, and the dynamic change of [Ca2+]i in podocytes was detected by a dynamic high-speed calcium imaging system. The results showed that TGF-β1 increased the protein expression of TRPC1/3/6 in podocytes, but had no effects on the protein expression of TRPC4. The protein expression levels of TRPC5/7 were only affected by 4 ng/mL and 8 ng/mL TGF-β1, respectively. TGF-β1 increased TRPC1/3/6 mRNA levels in podocytes, however had no effects on TRPC4/5/7 mRNA. TGF-β1 significantly increased [Ca2+]i in podocytes. Knockdown of TRPC1/4/5/7 in podocytes had no significant effect on the [Ca2+]i induced by TGF-β1, but TRPC3/6 knockdown significantly decreased the [Ca2+]i. There was no significant difference in the [Ca2+]i between the TRPC6 siRNA-treated group and SKF96365-treated group, but the [Ca2+]i of the TRPC3 siRNA-treated group was significantly higher than that of SKF96365-treated group. These results demonstrate that TGF-β1 increases the expression of the TRPC1/3/6 in podocytes. TGF-β1 increases [Ca2+]i in podocytes, which is dependent on the TRPC3/6 expression. Our results also suggest that the effect of TRPC6 on [Ca2+]i in podocytes may be greater than that of TRPC3.

Role and mechanism of hydrogen sulfide in vascular calcification / 生理学报

Vascular calcification is a common pathological process in patients with diabetes, chronic kidney disease, and cardiovascular disease, manifested by the deposition of hydroxyapatite on the walls of blood vessels. Hydrogen sulfide is the third gas signal molecule found in mammals after nitric oxide and carbon monoxide, which has anti-inflammatory, antioxidant stress and other effects in the cardiovascular system. In recent years, it has been recognized that hydrogen sulfide has an anti-vascular calcification effect, and supplementation with hydrogen sulfide and its donors can alleviate vascular calcification. In this review, we discussed the various evidence of the protective effect of hydrogen sulfide on vascular calcification, and highlighted the hydrogen sulfide metabolism changes and the potential regulatory mechanisms of hydrogen sulfide on the pathophysiological changes in vascular calcification.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

BACKGROUND: Designed mimetic molecules are attractive tools in biopharmaceuticals and synthetic biology. They require mass and functional production for the assessment of upcoming challenges in the near future. The DARPin family is considered a mimetic pharmaceutical peptide group with high affinity binding to specific targets. DARPin G3 is designed to bind to the HER2 (human epidermal growth factor receptor 2) tyrosine kinase receptor. Overexpression of HER2 is common in some cancers, including breast cancer, and can be used as a prognostic and predictive tool for cancer. The chloroplasts are cost-effective alternatives, equal to, and sometimes better than, bacterial, yeast, or mammalian expression systems. This research examined the possibility of the production of the first antibody mimetic, DARPin G3, in tobacco chloroplasts for HER2 imaging in oncology. RESULTS: The chloroplast specific DARPin G3 expression cassette was constructed and transformed into N. tabacum chloroplasts. PCR and Southern blot analysis confirmed integration of transgenes as well as chloroplastic and cellular homoplasmy. The Western blot analysis and ELISA confirmed the production of DARPin G3 at the commercial scale and high dose with the rate of 20.2% in leaf TSP and 33.7% in chloroplast TSP. The functional analysis by ELISA confirmed the binding of IMAC purified chloroplast-made DARPin G3 to the extracellular domain of the HER2 receptor with highly effective picomolar affinities. The carcinoma cellular studies by flow cytometry and immunofluorescence microscopy confirmed the correct functioning by the specific binding of the chloroplast-made DARPin G3 to the HER2 receptor on the surface of HER2-positive cancer cell lines. CONCLUSION: The efficient functional bioactive production of DARPin G3 in chloroplasts led us to introduce plant chloroplasts as the site of efficient production of the first antibody mimetic molecules. This report, as the first case of the cost-effective production of mimetic molecules, enables researchers in pharmaceuticals, synthetic biology, and bio-molecular engineering to develop tool boxes by producing new molecular substitutes for diverse purposes.

Apoptotic and autophagic death union by Thuja occidentalis homeopathic drug in cervical cancer cells with thujone as the bioactive principle / 中西医结合学报

OBJECTIVE@#"Multi-targeting" drugs can prove fruitful to combat drug-resistance of multifactorial disease-cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts.@*METHODS@#Thuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency.@*RESULTS@#Thuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC50) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways-apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC50 of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood-brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk.@*CONCLUSION@#Robust "multi-targeting" anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.

Prestin-Mediated Frequency Selectivity Does not Cover Ultrahigh Frequencies in Mice / 神经科学通报·英文版

In mammals, the piezoelectric protein, Prestin, endows the outer hair cells (OHCs) with electromotility (eM), which confers the capacity to change cellular length in response to alterations in membrane potential. Together with basilar membrane resonance and possible stereociliary motility, Prestin-based OHC eM lays the foundation for enhancing cochlear sensitivity and frequency selectivity. However, it remains debatable whether Prestin contributes to ultrahigh-frequency hearing due to the intrinsic nature of the cell's low-pass features. The low-pass property of mouse OHC eM is based on the finding that eM magnitude dissipates within the frequency bandwidth of human speech. In this study, we examined the role of Prestin in sensing broad-range frequencies (4-80 kHz) in mice that use ultrasonic hearing and vocalization (to >100 kHz) for social communication. The audiometric measurements in mice showed that ablation of Prestin did not abolish hearing at frequencies >40 kHz. Acoustic associative behavior tests confirmed that Prestin-knockout mice can learn ultrahigh-frequency sound-coupled tasks, similar to control mice. Ex vivo cochlear Ca2+ imaging experiments demonstrated that without Prestin, the OHCs still exhibit ultrahigh-frequency transduction, which in contrast, can be abolished by a universal cation channel blocker, Gadolinium. In vivo salicylate treatment disrupts hearing at frequencies <40 kHz but not ultrahigh-frequency hearing. By pharmacogenetic manipulation, we showed that specific ablation of the OHCs largely abolished hearing at frequencies >40 kHz. These findings demonstrate that cochlear OHCs are the target cells that support ultrahigh-frequency transduction, which does not require Prestin.

Detection, isolation, and characterization of a novel impurity from several folic acid products / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Folic acid belongs to the group of water-soluble B vitamins and naturally exists in multiple forms in a wide variety of foods such as legumes, vegetables, liver, and milk (Iyer and Tomar, 2009; Lyon et al., 2020). It is involved in many biochemical reactions critical for cell division, such as purine and pyrimidine biosynthesis, DNA/RNA biosynthesis, and amino acid metabolism (Iyer and Tomar, 2009). Mammals cannot synthesize folic acid and thus they must acquire it from food. Although folic acid is ubiquitous in foods, folic acid deficiency still often occurs due to various causes such as unhealthy diet (Hildebrand et al., 2021; Iimura et al., 2022), disease-related malabsorption (Arcot and Shrestha, 2005), medication-related depletion (Arcot and Shrestha, 2005), or vitamin B12 deficiency (Fishman et al., 2000). Folic acid deficiency has been associated with several health problems, such as anemia (Carmel, 2005; Bailey and Caudill, 2012), cancer (Duthie, 1999), cardiovascular diseases (Wald et al., 2002), neural tube defects in newborns (van der Put et al., 2001), neuropsychiatric dysfunction (Shea et al., 2002), depression (Falade et al., 2021), inflammatory diseases (Suzuki and Kunisawa, 2015; Jones et al., 2019), and eye diseases (Sijilmassi, 2019). To prevent folic acid deficiency, its daily intake (400 μg/d) has been recommended for adults in the European Union, and its increased intake (600 μg/d) is advised for women before and during pregnancy (FAO/WHO, 2002; IOM, 2004). The New Zealand government mandated the fortification of non-organic wheat flour with folic acid in July 2021, and the UK government mandated the fortification of non-wholemeal wheat flour with folic acid in September 2021 (Haggarty, 2021).