Extracellular pectin-RALF phase separation mediates FERONIA global signaling function.

The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.

Immunologic, Anti-Inflammatory, and Anti-Muscle Damage Profile of Supplemented Vitamin D3 in Healthy Adults on Strenuous Endurance Exercise.

Reportedly, strenuous endurance exercise can depress the immune system and induce inflammation and muscle damage. Therefore, this double-blinded, matched-pair study aimed to investigate the impact of vitamin D3 supplementation on immune response (leukocyte, neutrophil, lymphocyte, CD4+, CD8+, CD19+, and CD56+ counts), inflammatory profile (TNF-α and IL-6), muscle damage (CK and LDH levels), as well as aerobic capacity after strenuous endurance exercise in 18 healthy men taking 5000 IU of vitamin D3 (n = 9) or placebo (n = 9) daily for 4 weeks. Total and differential blood leukocyte counts, levels of cytokines, and muscle damage biomarkers were determined before, immediately after, and 2, 4, and 24 h after exercise. The IL-6, CK, and LDH levels were significantly lower in vitamin D3 group at 2, 4, and 24 h post exercise (p < 0.05). Maximal and average heart rates during exercise were also significantly lower (p < 0.05). In the vitamin D3 group, the CD4+/CD8+ ratio after 4 weeks of supplementation was only significantly lower at post-0 than at baseline and significantly higher at post-2 than at baseline and post-0 (all p < 0.05). Taken together, 5000 IU of daily vitamin D3 supplementation for 4 weeks exhibited positive effects in terms of increased blood 25(OH)D levels, CD4+/CD8+ ratio (immune response), and aerobic capacity while inhibiting inflammatory cytokines and CK and LDH (muscle damage) in people performing strenuous endurance exercise.

Pollen-pistil interactions: It takes two to tangle but a molecular cast of many to deliver.

Explosive advances have been made in the molecular understanding of pollen-pistil interactions that underlie reproductive success in flowering plants in the past three decades. Among the most notable is the discovery of pollen tube attractants [1∗,2∗]. The roles these molecules play in facilitating conspecific precedence thus promoting interspecific genetic isolation are also emerging [3-5]. Male-female interactions during the prezygotic phase and contributions from the male and female gametophytes have been comprehensively reviewed recently. Here, we focus on key advances in understanding the mechanistic underpinnings of how these interactions overcome barriers at various pollen-pistil interfaces along the pollen tube growth pathway to facilitate fertilization by desirable mates.

Non-Muscular Invasive Bladder Cancer: Re-envisioning Therapeutic Journey from Traditional to Regenerative Interventions.

Non-muscular invasive bladder cancer (NMIBC) is one of the most common cancer and major cause of economical and health burden in developed countries. Progression of NMIBC has been characterized as low-grade (Ta) and high grade (carcinoma in situ and T1). The current surgical intervention for NMIBC includes transurethral resection of bladder tumor; however, its recurrence still remains a challenge. The BCG-based immunotherapy is much effective against low-grade NMIBC. BCG increases the influx of T cells at bladder cancer site and inhibits proliferation of bladder cancer cells. The chemotherapy is another traditional approach to address NMIBC by supplementing BCG. Notwithstanding, these current therapeutic measures possess limited efficacy in controlling NMIBC, and do not provide comprehensive long-term relief. Hence, biomaterials and scaffolds seem an effective medium to deliver therapeutic agents for restructuring bladder post-treatment. The regenerative therapies such as stem cells and PRP have also been explored for possible solution to NMIBC. Based on above-mentioned approaches, we have comprehensively analyzed therapeutic journey from traditional to regenerative interventions for the treatment of NMIBC.

Low-frequency electroacupuncture at acupoints guanyuan (CV4) and zhongji (CV3) lengthen ejaculatory latency and improves sexual behavior in male rats.

Acupuncture is a key component of Traditional Chinese Medicine in which needles are inserted into specific areas in the body to stimulate certain physiological reactions of the body. Clinical research shows that acupuncture is beneficial in treating a variety of illnesses, such as erectile dysfunction. Therefore, we investigated the effect of electroacupuncture (EA) stimulation of both low- and high frequencies at Guanyuan (CV4) and Zhongji (CV3) acupoints on male copulatory behavior in sexually experienced male rats. The animals were randomly divided into four groups: control, sham EA, EA, and only acupuncture. The administered low and high EA were 2 Hz and 80 Hz, respectively, for which the respective intensities were 1.5 mA (30 min/day for 5 days) and 1.5 mA (30 min once). The only acupuncture-administered group received acupunctures at CV4 and CV3 without electrical stimulation. Following the acupuncture treatments, copulatory behavior test was conducted. Eventually, animals were sacrificed and blood samples were collected for testing the serum hormonal profile including luteinizing hormone (LH), testosterone (T), and serotonin (5-HT). Results showed that low-frequency EA-treated rats exhibited increasing intromissions and ejaculation latency compared to control, sham EA, and acupuncture groups, while high-frequency EA-treated rats displayed lower intromissions and ejaculation frequency when compared with those in controls. Furthermore, serum levels of 5-HT and LH in low-frequency EA-treated rats were higher than all the other groups. These results indicate that compared to high-frequency EA, the low-frequency EA might be efficacious in the treatment of premature ejaculation, thereby improving sexual behavior.

Ovatodiolide, isolated from Anisomeles indica, suppresses bladder carcinogenesis through suppression of mTOR/ß-catenin/CDK6 and exosomal miR-21 derived from M2 tumor-associated macrophages.

Bladder cancer (BCa) is the fourth leading cause of cancer deaths worldwide due to its aggressiveness and resistance against therapies. Intricate interactions between cancer cells and the tumor microenvironment (TME) are essential for both disease progression and regression. Thus, interrupting molecular communications within the TME could potentially provide improved therapeutic efficacies. M2-polarized tumor-associated macrophages (M2 TAMs) were shown to contribute to BCa progression and drug resistance. We attempted to provide evidence for ovatodiolide (OV) as a potential therapeutic agent that targets both TME and BCa cells. First, tumor-suppressing functions of OV were determined by cell viability, colony, and tumor-sphere formation assays using a coculture system composed of M2 TAMs/BCa cells. Subsequently, we demonstrated that extracellular vesicles (EVs) isolated from M2 TAMs containing oncomiR-21 and mRNAs, including Akt, STAT3, mTOR, and ß-catenin, promoted cisplatin (CDDP) resistance, migration, and tumor-sphere generation in BCa cells, through increasing CDK6, mTOR, STAT3, and ß-catenin expression. OV treatment also prevented M2 polarization and reduced EV cargos from M2 TAMs. Finally, in vivo data demonstrated that OV treatment overcame CDDP resistance. OV only and the OV + CDDP combination both resulted in significant reductions in mTOR, ß-catenin, CDK6, and miR-21 expression in tumor samples and EVs isolated from serum. Collectively, we demonstrated that M2 TAMs induced malignant properties in BCa cells, in part via oncogenic EVs. OV treatment prevented M2 TAM polarization, reduced EV cargos derived from M2 TAMs, and suppressed ß-catenin/mTOR/CDK6 signaling. These findings provide preclinical evidence for OV as a single or adjuvant agent for treating drug-resistant BCa.

FERONIA controls pectin- and nitric oxide-mediated male-female interaction.

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release4. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.

Cordyceps militaris Treatment Preserves Renal Function in Type 2 Diabetic Nephropathy Mice.

Diabetic nephropathy is derived from long-term effects of high blood glucose on kidney function in type 2 diabetic patients. Several antidiabetic drugs and herbal medications have failed to prevent episodes of DN. Hence, this study aimed to further investigate the renal injury-reducing effect of antidiabetic CmNo1, a novel combination of powders of fruiting bodies and mycelia of Cordyceps militaris. After being administered with streptozotocin-nicotinamide and high-fat-diet, the diabetic nephropathy mouse model displayed elevated blood glucose and renal dysfunction markers including serum creatinine and kidney-to-body weight ratio. These elevated markers were significantly mitigated following 8 weeks CmNo1 treatment. Moreover, the chronic hyperglycemia-induced pathological alteration in renal tissue were also ameliorated. Besides, immunohistochemical study demonstrated a substantial reduction in elevated levels of carboxymethyl lysine, an advanced glycation end product. Elevated collagenous deposition in DN group was also attenuated through CmNo1 administration. Moreover, the enhanced levels of transforming growth factor-ß1, a fibrosis-inducing protein in glomerulus were also markedly dampened. Furthermore, auxiliary risk factors in DN like serum triglycerides and cholesterol were found to be increased but were decreased by CmNo1 treatment. Conclusively, the results suggests that CmNo1 exhibit potent and efficacious renoprotective action against hyperglycemia-induced DN.

A novel lily anther-specific gene encodes adhesin-like proteins associated with exine formation during anther development.

The anther-specific gene LLA1271 isolated from lily (Lilium longiflorum Thunb.) anthers is novel and exists in two forms. The protein encoded by LLA1271 may represent an adhesin-like protein first found in higher plants. The protein contains a typical N-terminal signal peptide followed by a highly conserved repeat domain. The LLA1271 gene is temporally expressed at the phase of microspore development. RNA blot and RNA in situ hybridization analyses demonstrated that the gene was expressed both in the tapetum and in the microspore. The gene is endo- and exogenously induced by gibberellin. Studies with the gibberellin biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that LLA1271 is negatively regulated by ethylene, and a cross-talk of regulation between gibberellin and ethylene occurs in young anthers. The treatment with NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development in a state close to that of a tapetum without treatment. The LLA1271 protein is heat stable and heterogeneous. An immunoblot of separated protein fractions of the anther revealed that the LLA1271 protein was detected in protein fraction of the microspore released from the cell wall by treatment with either 0.5% or 2% Triton X-100. Ectopic expression of LLA1271 resulted in impaired stamen and low pollen germination. Scanning electron microscopy of TAP::LLA1271 pollen showed distorted exine formation and patterning. The LLA1271 protein once synthesized in both the tapetum and microspore is secreted and deposited on the surface of microspores, moderately affecting exine formation and patterning.

Identification of the tapetum/microspore-specific promoter of the pathogenesis-related 10 gene and its regulation in the anther of Lilium longiflorum.

A tapetum/microspore-specific pathogenesis-related (PR) 10 gene was previously identified in lily (Lilium longiflorum Thunb.) anthers. In situ hybridization and RNA blot analysis indicated that the lily PR10 genes are expressed specifically and differentially in the tapetum of the anther wall and in microspores during anther development. The accumulation of PR10 transcripts was exogenously induced by gibberellic acid (GA) and was suppressed by ethylene. Studies using inhibitors of GA and ethylene revealed that the lily PR10 is modulated by an antagonistic interaction between GA and ethylene. The treatment of norbornadien, an ethylene inhibitor, caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole, an inhibitor of GA biosynthesis, arrested tapetal development to a status close to that of control. The expression of the lily PR10g promoter in transgenic Arabidopsis was determined using the ß-glucuronidase (GUS) reporter gene indicated that the decisive fragment required for anther specificity is located -1183 bp to -880 bp upstream of the transcription start site. The PR10gPro::barnase transgenic lines exhibited complete male sterility because of the disruption of the tapetum and the deformation of microspore/pollen. The anther specificity of lily PR10 highlights the importance of the tapetum/microspore-specific PR10g promoter for future biotechnological and agricultural applications.

Potential Osteoporosis Recovery by Deep Sea Water through Bone Regeneration in SAMP8 Mice.

The aim of this study is to examine the therapeutic potential of deep sea water (DSW) on osteoporosis. Previously, we have established the ovariectomized senescence-accelerated mice (OVX-SAMP8) and demonstrated strong recovery of osteoporosis by stem cell and platelet-rich plasma (PRP). Deep sea water at hardness (HD) 1000 showed significant increase in proliferation of osteoblastic cell (MC3T3) by MTT assay. For in vivo animal study, bone mineral density (BMD) was strongly enhanced followed by the significantly increased trabecular numbers through micro-CT examination after a 4-month deep sea water treatment, and biochemistry analysis showed that serum alkaline phosphatase (ALP) activity was decreased. For stage-specific osteogenesis, bone marrow-derived stromal cells (BMSCs) were harvested and examined. Deep sea water-treated BMSCs showed stronger osteogenic differentiation such as BMP2, RUNX2, OPN, and OCN, and enhanced colony forming abilities, compared to the control group. Interestingly, most untreated OVX-SAMP8 mice died around 10 months; however, approximately 57% of DSW-treated groups lived up to 16.6 months, a life expectancy similar to the previously reported life expectancy for SAMR1 24 months. The results demonstrated the regenerative potentials of deep sea water on osteogenesis, showing that deep sea water could potentially be applied in osteoporosis therapy as a complementary and alternative medicine (CAM).

Expression, localization and function of a cis-prenyltransferase in the tapetum and microspores of lily anthers.

The cis-prenyltransferase gene LLA66 (Lilium longiflorum anther-66), the first prenyltransferase to be identified in the tapetum and microspores, was selected from a suppression subtractive cDNA library during microspore development in the anther of L. longiflorum. The LLA66 cDNA encodes a polypeptide of 308 amino acids with a calculated molecular mass of 35.7 kDa. Thermal asymmetric interlaced-PCR was employed to obtain the 5'-regulatory region of LLA66. Sequence alignment revealed that the LLA66 protein shares 30-41% identity with cis-prenyltransferases of various broad-spectrum species and is phylogenetically distinct from other monocot cis-prenyltransferases. Based on critical regulatory domains in cis-prenyltransferase, LLA66 was concluded to catalyze the production of long-chain polyprenyl products. RNA blot analysis indicated that the LLA66 gene is anther specific and differentially expressed during microspore development in the anther. In situ hybridization with the digoxigenin-labeled antisense riboprobe of LLA66 showed strong signals at the tapetal layer of the anther wall. The LLA66 mRNA was also coordinately detected in the microspores. Furthermore, gibberellin inhibitor analysis indicated that the LLA66 gene is endogenously induced by gibberellin, but its induction is independent of ethylene regulation. Reverse transcription-PCR analysis indicated that gene expression of LLA66 both in the microspore and in the anther wall increased to the maximum level, at which stage the tapetum became highly active and secretory. The enzyme activity of prenyltransferases in various stages of microspore development correlated with tapetal growth and disintegration. LLA66 was introduced into Saccharomyces cerevisiae, and the His-tagged LLA66 protein was affinity purified using Ni(2+)-nitrilotriacetic acid-agarose. The involvement of cis-prenyltransferase in the anther in the synthesis of dolichols and polyprenols is discussed.

Expression and regulation of two novel anther-specific genes in Lilium longiflorum.

Two stage-specific genes have been isolated from a subtractive cDNA library constructed from developing anthers of lily (Lilium longiflorum). The proteins encoded by the two genes have a strong hydrophobic region at the N-terminus, indicating the presence of a signal peptide. The deduced LLA-67 is a new type of small cysteine-rich protein whose sequence exhibits four consecutive CX(3)CX(6-10) repeats that could form signal-receiving finger motifs, while the deduced LLA-115 protein shows significant similarities to a rice unknown protein, and putative cell wall proteins of Medicago truncatula and Arabidopsis. The transcripts of LLA-67 and LLA-115 were anther specific and differentially detected at the phase of microspore development. In situ hybridization with antisense riboprobes of the two genes in the anther showed strong signals localized to the tapetal layer of the anther wall. The LLA-67 mRNA was also detected in the microspore at the phase of microspore development but the LLA-115 mRNA was not. The LLA-115 gene can be exogenously induced by gibberellin (GA), whereas the LLA-67 gene cannot be induced. Studies with the GA biosynthesis inhibitor uniconazole and an inhibitor of ethylene activity, 2,5-norbornadien (NBD), revealed that the two genes were negatively regulated by ethylene and a cross-talk between GA and ethylene was involved in the regulation of the two genes occurring in young anthers. The treatment of NBD caused the tapetum to become densely cytoplasmic and highly polarized, whereas uniconazole arrested tapetal development to a status close to that of control. DNA blots of lily genomic DNA indicated that the two genes were encoded by a small gene family. The different actions of hormones on gene expression and the possible function of the gene products in young anthers are discussed.