Utilizing the allyl-terminated copolymer methoxy(poly(ethylene glycol))-block-poly(jasmine lactone) in the development of amorphous solid dispersions: A comparative study of functionalized and non-functionalized polymer.

Molecular interactions are crucial to stabilize amorphous drugs in amorphous solid dispersions (ASDs). Most polymers, however, have only a limited ability to form strong molecular interactions with drugs. Polymers tailored to fit the physicochemical properties of the drug molecule to be incorporated, for instance by allowing the incorporation of specific functional groups, would be highly sought-for in this regard. For this purpose, the novel allyl-terminated polymer methoxy(polyethylene glycol)-block-poly(jasmine lactone) (mPEG-b-PJL) has been synthesized and functionalized to potentially enhance specific drug-polymer interactions. This study investigated the use of mPEG-b-PJL in ASDs, using carvedilol (CAR), a weakly basic model drug. The findings revealed that the acidic functionalized form of the polymer (mPEG-b-PJL-COOH) indeed established stronger molecular interactions with CAR compared to its non-functionalized counterpart mPEG-b-PJL. Evaluations on polymer effectiveness in forming ASDs demonstrated that mPEG-b-PJL-COOH outperformed its non-functionalized counterpart in miscibility, drug loading ability, and stability, inferred from reduced molecular mobility. However, dissolution tests indicated that ASDs with mPEG-b-PJL-COOH did not significantly improve the dissolution behaviour compared to amorphous CAR alone, despite potential solubility enhancement through micelle formation. Overall, this study confirms the potential of functionalized polymers in ASD formulations, while the challenge of improving dissolution performance in these ASDs remains an area of further development.

A cyclic peptide-based PROTAC induces intracellular degradation of palmitoyltransferase and potently decreases PD-L1 expression in human cervical cancer cells.

Introduction: Our previous research has found that degradation of palmitoyltransferase in tumor cells using a linear peptide PROTAC leads to a significant decrease in PD-L1 expression in tumors. However, this degradation is not a sustained and efficient process. Therefore, we designed a cyclic peptide PROTAC to achieve this efficient anti-PD-L1 effect. Methods: We designed and synthesized an improvement in linear peptide PROTAC targeting palmitoyltransferase DHHC3, and used disulfide bonds to stabilize the continuous N- and C-termini of the peptides to maintain their structure. Cellular and molecular biology techniques were used to test the effect of this cyclic peptide on PD-L1. Results: In human cervical cancer cells, our cyclic peptide PROTAC can significantly downregulate palmitoyl transferase DHHC3 and PD-L1 expressions. This targeted degradation effect is enhanced with increasing doses and treatment duration, with a DC50 value much lower than that of linear peptides. Additionally, flow cytometry analysis of fluorescence intensity shows an increase in the amount of cyclic peptide entering the cell membrane with prolonged treatment time and higher concentrations. The Cellular Thermal Shift Assay (CETSA) method used in this study indicates effective binding between our novel cyclic peptide and DHHC3 protein, leading to a change in the thermal stability of the latter. The degradation of PD-L1 can be effectively blocked by the proteasome inhibitor MG132. Results from clone formation experiments illustrate that our cyclic peptide can enhance the proliferative inhibition effect of cisplatin on the C33A cell line. Furthermore, in the T cell-C33A co-culture system, cyclic peptides target the degradation of PD-L1, thereby blocking the interaction between PD-L1 and PD-1, and promoting the secretion of IFN-γ and TNF-α in the co-culture system supernatant. Conclusion: Our results demonstrate that a disulfide-bridged cyclic peptide PROTAC targeting palmitoyltransferase can provide a stable and improved anti-PD-L1 activity in human tumor cells.

Ethylene insensitive 2 (EIN2) as a potential target gene to enhance Fusarium head blight disease resistance.

Fusarium head blight (FHB) caused by Fusarium graminearum (Fg) severely affects cereal crops, especially wheat and barley. FHB results in significant yield loss, reduces grain quality and contaminates grains with mycotoxin. The development of FHB-resistant cereal cultivars can be expedited through CRISPR gene editing. The Arabidopsis ethylene insensitive 2 (AtEIN2) plays a key role in ethylene signaling pathway and is critical for monitoring plant growth and defense responses. RNAi down-regulation of the wheat homolog TaEIN2 has been shown to enhance wheat FHB resistance. Here we generated site-specific mutations in AtEIN2 by CRISPR-editing. Detached inflorescence infection assays revealed that AtEIN2 knock-out (KO) mutants displayed enhanced Fg resistance and substantially reduced Fg spore production in planta. Gene expression profiling of defense genes revealed that impairment of AtEIN2 resulted in down-regulation of the ethylene signaling pathway while the salicylic acid signaling pathway was unaffected. Complementation of AtEIN2-KO plants with a barley orthologue, HvEIN2, restored Fg susceptibility, indicating that HvEIN2 is functionally equivalent to its Arabidopsis counterpart and, hence, may have a similar role in conditioning barley Fg susceptibility. These results provide insight into the defense role of EIN2 and a molecular and functional foundation for manipulating HvEIN2 to enhance FHB resistance in barley.

Parafoveal retinal massage combined with autologous blood cover in the management of giant, persistent or recurrent macular holes.

AIM: To assess the efficacy and safety of parafoveal retinal massage combined with autologous whole blood cover in the treatment of refractory macular holes (MHs) and present the surgical procedure. METHODS: Patients with giant (minimum diameter ≥800 µm), recurrent or persistent MHs who underwent PPV combined with parafoveal retinal massage and autologous whole blood cover using C3F8 as tamponade agent from February 2018 to May 2019 were enrolled in this retrospective study. After surgery, all patients were informed to maintain a prone position for at least 7d. Preoperative and postoperative best-corrected visual acuities (BCVAs) were compared and MH closure rate was measured as the main outcome. RESULTS: A total of 13 MH patients consisted of 6 giant MHs, 4 persistent holes and 3 recurrent holes (5 men and 8 women; average age was 56.40±11.72y) were enrolled in this study. MH closure was achieved in 11 eyes by this modified surgical technique while 2 eyes failed. Revitrectomy with autologous neurosensory retinal patch transplantations was applied for those 2 patients and then both holes were closed. No intraoperative complications were observed. BCVA improved from 1.73 logMAR to 0.74 logMAR at 6mo postoperation. There was significant difference in BCVA before versus after the surgery (P<0.05). There were no adverse events occurred during the follow-up period. CONCLUSION: With easier surgical procedure, parafoveal retinal massage combined with autologous whole blood cover is an effective addition to the surgical options for the management of refractory MHs.

Metformin Corrects Abnormal Circadian Rhythm and Kir4.1 Channels in Diabetes.

Purpose: Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5' adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice. Methods: Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel. Results: The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression. Conclusions: Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR.

Detection of human papillomavirus in squamous cell carcinoma arising from dermoid cysts.

OBJECTIVE: Primary squamous cell carcinoma (SCC) of the ovary in humans is rare. Most cases represent a malignant transformation of ovarian teratoma, Brenner tumor, or endometriosis. The etiology of this cancer remains largely unknown. Human papillomavirus (HPV) infection is a critical factor that induces tumor formation, particularly cervical cancer. Therefore, this study aimed to evaluate the association of HPV with malignant transformation of mature cystic teratoma (MCT) into SCC of the ovary. MATERIALS AND METHODS: The samples included four formalin-fixed paraffin-embedded SCC-MCT tissues and their adjacent tissues from the cervix to the ovaries, 12 cases of benign teratoma ovarian tissues (dermoid tissues), and 11 cases of benign nonteratoma ovarian tissues (nondermoid tissues). The two squamous carcinoma tissues of the cervix were used as control samples. HPV was detected by immunohistochemistry (IHC) with anti-HPV capsid or E6 (HPV type 16/18) antibodies and in situ hybridization (ISH) with three sets of genotyping probes, HPV types 6/11, 16/18, and 31/33. RESULTS: IHC revealed HPV infection associated with the four cases of SCC-MCT and the two cases of control cervical cancer samples. Importantly, HPV was also detected in adjacent reproductive tissues of the SCC-MCT cases, which suggested that the viral particles might spread in an ascending route through the fallopian tubes, endometrium, endocervix, and cervix to the ovaries. ISH revealed HPV type 16/18 in all SCC-MCT cases and HPV type 31/33 in two, with no HPV type 6/11 in any SCC-MCT cases. However, compared with the SCC-MCT cases, the lower detection rates of HPV in dermoid cysts and nondermoid tissues suggested that HPV might not be associated with normal ovarian tissues or benign ovarian teratomas. CONCLUSION: Our data suggest that high-risk HPV infection might be a causal factor that induces malignant transformation of MCT into SCC of the ovary, although further investigation is still required.

Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells.

Cranberry juice has been recognized as a treatment for urinary tract infections on the basis of scientific reports of proanthocyanidin anti-adhesion activity against Escherichia coli as well as from folklore. Xyloglucan oligosaccharides were detected in cranberry juice and the residue remaining following commercial juice extraction that included pectinase maceration of the pulp. A novel xyloglucan was detected through tandem mass spectrometry analysis of an ion at m/z 1055 that was determined to be a branched, three hexose, four pentose oligosaccharide consistent with an arabino-xyloglucan structure. Two-dimensional nuclear magnetic resonance spectroscopy analysis provided through-bond correlations for the α-L-Araf (1→2) α-D-Xylp (1→6) ß-D-Glcp sequence, proving the S-type cranberry xyloglucan structure. Cranberry xyloglucan-rich fractions inhibited the adhesion of E. coli CFT073 and UTI89 strains to T24 human bladder epithelial cells and that of E. coli O157:H7 to HT29 human colonic epithelial cells. SSGG xyloglucan oligosaccharides represent a new cranberry bioactive component with E. coli anti-adhesion activity and high affinity for type 1 fimbriae.

An N-terminal fragment of yeast ribosomal protein L3 inhibits the cytotoxicity of pokeweed antiviral protein in Saccharomyces cerevisiae.

We have previously shown that ribosomal protein L3 is required for pokeweed antiviral protein (PAP), a type I ribosome inactivating protein, to bind to ribosomes and depurinate the α-sarcin/ricin loop (SRL) in yeast. Co-expression of the N-terminal 99 amino acids of yeast L3 (L3Δ99) with PAP in transgenic tobacco plants completely abolished the toxicity of PAP. In this study, we investigated the interaction between PAP and L3Δ99 in Saccharomyces cerevisiae. Yeast cells co-transformed with PAP and L3Δ99 showed markedly reduced growth inhibition and reduced rRNA depurination by PAP, compared to cells transformed with PAP alone. Co-transformation of yeast with PAP and L3Δ21 corresponding to the highly conserved N-terminal 21 amino acids of L3Δ99, reduced the cytotoxicity of PAP. PAP mRNA and protein levels were elevated and L3Δ99 or L3Δ21 mRNA and protein levels were reduced in yeast co-transformed with PAP and L3Δ99 or with PAP and L3Δ21, respectively. PAP interacted with L3Δ21 in yeast cells in vivo and by Biacore analysis in vitro, suggesting that the interaction between L3Δ21 and PAP may inhibit PAP-mediated depurination of the SRL, leading to a reduction in the cytotoxicity of PAP.

Biodegradable ferulic acid-containing poly(anhydride-ester): degradation products with controlled release and sustained antioxidant activity.

Ferulic acid (FA) is an antioxidant and photoprotective agent used in biomedical and cosmetic formulations to prevent skin cancer and senescence. Although FA exhibits numerous health benefits, physicochemical instability leading to decomposition hinders its efficacy. To minimize inherent decomposition, a FA-containing biodegradable polymer was prepared via solution polymerization to chemically incorporate FA into a poly(anhydride-ester). The polymer was characterized using nuclear magnetic resonance and infrared spectroscopies. The molecular weight and thermal properties were also determined. In vitro studies demonstrated that the polymer was hydrolytically degradable, thus providing controlled release of the chemically incorporated bioactive with no detectable decomposition. The polymer degradation products were found to exhibit antioxidant and antibacterial activity comparable to that of free FA, and in vitro cell viability studies demonstrated that the polymer is noncytotoxic toward fibroblasts. This renders the polymer a potential candidate for use as a controlled release system for skin care formulations.

Fast speciation of mercury in seawater by short-column high-performance liquid chromatography hyphenated to inductively coupled plasma spectrometry after on-line cation exchange column preconcentration.

A simple and fast method for trace speciation analysis of mercury (Hg(2+)), methylmercury (MeHg(+)) and ethylmercury (EtHg(+)) in seawater has been developed by short-column high-performance liquid chromatography hyphenated to inductively coupled plasma spectrometry (HPLC-ICP-MS) after on-line cation-exchange column (CEC) preconcentration. The analytes were firstly adsorbed on the CEC without any extraneous reagent, and then were eluted rapidly (within seconds) and completely with a very low concentration of l-cysteine solution, which provides the conveniency for the on-line coupling of the preconcentration method and detection technique. To our best knowledge, it is for the first time to employ the CEC preconcentration technique to trap all of the three mercury species simultaneously at their positive charged status for the purpose of speciation analysis. Under the optimized conditions, a very high preconcentration factor up to 1250 has been obtained with 30mL sample solution, which leads to the very low detection limits of 0.042ngL(-1) for Hg(2+), 0.016ngL(-1) for MeHg(+) and 0.008ngL(-1) for EtHg(+) (as Hg), respectively. With the established method, three seawater samples were also analyzed, and all the three mercury species have been found in each sample, albeit at a very low concentration.

Anti-inflammatory activities of mogrosides from Momordica grosvenori in murine macrophages and a murine ear edema model.

Momordica grosvenori (Luo Han Guo), grown primarily in Guangxi province in China, has been traditionally used for thousands of years by the Chinese to make hot drinks for the treatment of sore throat and the removal of phlegm. The natural noncaloric sweetening triterpenoid glycosides (mogrosides) contained in the M. grosvenori fruits are also antioxidative, anticarcinogenic, and helpful in preventing diabetic complications. The aim of this study was to assess the anti-inflammatory properties of mogrosides in both murine macrophage RAW 264.7 cells and a murine ear edema model. The results indicate that mogrosides can inhibit inflammation induced by lipopolysaccharides (LPS) in RAW 264.7 cells by down-regulating the expression of key inflammatory genes iNOS, COX-2, and IL-6 and up-regulating some inflammation protective genes such as PARP1, BCL2l1, TRP53, and MAPK9. Similarly, in the murine ear edema model, 12-O-tetradecanoylphorbol-13-acetate-induced inflammation was inhibited by mogrosides by down-regulating COX-2 and IL-6 and up-regulating PARP1, BCL2l1, TRP53, MAPK9, and PPARδ gene expression. This study shows that the anticancer and antidiabetic effects of M. grosvenori may result in part from its anti-inflammatory activity.

Small-molecule inhibitor leads of ribosome-inactivating proteins developed using the doorstop approach.

Ribosome-inactivating proteins (RIPs) are toxic because they bind to 28S rRNA and depurinate a specific adenine residue from the α-sarcin/ricin loop (SRL), thereby inhibiting protein synthesis. Shiga-like toxins (Stx1 and Stx2), produced by Escherichia coli, are RIPs that cause outbreaks of foodborne diseases with significant morbidity and mortality. Ricin, produced by the castor bean plant, is another RIP lethal to mammals. Currently, no US Food and Drug Administration-approved vaccines nor therapeutics exist to protect against ricin, Shiga-like toxins, or other RIPs. Development of effective small-molecule RIP inhibitors as therapeutics is challenging because strong electrostatic interactions at the RIP•SRL interface make drug-like molecules ineffective in competing with the rRNA for binding to RIPs. Herein, we report small molecules that show up to 20% cell protection against ricin or Stx2 at a drug concentration of 300 nM. These molecules were discovered using the doorstop approach, a new approach to protein•polynucleotide inhibitors that identifies small molecules as doorstops to prevent an active-site residue of an RIP (e.g., Tyr80 of ricin or Tyr77 of Stx2) from adopting an active conformation thereby blocking the function of the protein rather than contenders in the competition for binding to the RIP. This work offers promising leads for developing RIP therapeutics. The results suggest that the doorstop approach might also be applicable in the development of other protein•polynucleotide inhibitors as antiviral agents such as inhibitors of the Z-DNA binding proteins in poxviruses. This work also calls for careful chemical and biological characterization of drug leads obtained from chemical screens to avoid the identification of irrelevant chemical structures and to avoid the interference caused by direct interactions between the chemicals being screened and the luciferase reporter used in screening assays.

Identification of amino acids critical for the cytotoxicity of Shiga toxin 1 and 2 in Saccharomyces cerevisiae.

Shiga toxins (Stx1 and Stx2) are produced by E. coli O157:H7, which is a leading cause of foodborne illness. The A subunits of Stx1 (Stx1A) and Stx2 (Stx2A) are ribosome inactivating proteins (RIPs) that inhibit translation by removing an adenine from the highly conserved α-sarcin ricin loop (SRL) of the large rRNA. Here, we used mutagenesis in Saccharomyces cerevisiae to identify residues critical for cytotoxicity of Stx1A and Stx2A. The A subunits depurinated the SRL, inhibited translation and caused apoptotic-like cell death in yeast. Single mutations in Asn75, Tyr77, Glu167 and Arg176 reduced the cytotoxicity of both toxins around 10-fold. However, Asn75 and Tyr77 were more critical for the depurination activity of Stx2A, while Arg176 was more critical for the depurination activity of Stx1A. The crystal structures of the two proteins lack electron density for some surface loops, including one which is adjacent to the active site in both molecules. Modeling these loops changed neither the secondary nor the tertiary structures of the rest of the protein. Analysis of solvent accessible surface areas indicated that Asn75 and Tyr77 are more exposed in Stx2A, while Arg176 is more exposed in Stx1A, indicating that residues with higher surface exposure were more critical for enzymatic activity. Double mutations at Glu167 and Arg176 eliminated the depurination activity and cytotoxicity of both toxins. C-terminal deletions of A chains eliminated cytotoxicity of both toxins, but showed functional differences. Unlike Stx1A, cytotoxicity of Stx2A was lost before its ability to depurinate ribosomes. These results identify residues that affect enzymatic activity and cytotoxicity of Stx1A and Stx2A differently and demonstrate that the function of these residues can be differentiated in yeast. The extent of ribosome depurination and translation inhibition did not correlate with the extent of cell death, indicating that depurination of the SRL and inhibition of translation are not entirely responsible for cell death.

Expression of a truncated form of ribosomal protein L3 confers resistance to pokeweed antiviral protein and the Fusarium mycotoxin deoxynivalenol.

The contamination of important agricultural products such as wheat, barley, or maize with the trichothecene mycotoxin deoxynivalenol (DON) due to infection with Fusarium species is a worldwide problem. Trichothecenes inhibit protein synthesis by targeting ribosomal protein L3. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein binds to L3 to depurinate the alpha-sarcin/loop of the large rRNA. Plants transformed with the wild-type PAP show lesions and express very low levels of PAP because PAP autoregulates its expression by destabilizing its own mRNA. We show here that transgenic tobacco plants expressing both the wild-type PAP and a truncated form of yeast L3 (L3delta) are phenotypically normal. PAP mRNA and protein levels are very high in these plants, indicating that L3delta suppresses the autoregulation of PAP mRNA expression. Ribosomes are not depurinated in the transgenic plants expressing PAP and L3delta, even though PAP is associated with ribosomes. The expression of the endogenous tobacco ribosomal protein L3 is up-regulated in these plants and they are resistant to the Fusarium mycotoxin DON. These results demonstrate that expression of an N-terminal fragment of yeast L3 leads to trans-dominant resistance to PAP and the trichothecene mycotoxin DON, providing evidence that both toxins target L3 by a common mechanism.

[Experimental and clinical study on functional restoration of the penis with large partial defect].

OBJECTIVE: To investigate the effective restoration method in penile partial defect. METHODS: To measure the thickness of suspensory ligaments on cadavers; To find the relationship between inferior pubic ramus and corpora cavernosa crus through corpora cavernosa angiography and ultrasonic scanning. The procedure of restoration was based on the use of dissection of the suspensory ligaments and part of crus, and the local fat flap had been transferred to fill the front space of symphysis ossium pubi. Various flaps were designed as coverage material. Penis residual stump was advanced to anterior portion of the newly restoration penile body as "glans"; To take full layer skin of penis from post-operating patients to observe neurofibril regeneration by light microscopy, electron microscopy and scanning electron microscopy; To determine sensation recovery by physical examination; To check erectile function with NEVA System. RESULTS: The whole suspensory ligaments thickness was 7.66 cm in average on 30 cadavers (60 sides). The crus were attached to bilateral inferior pubic ramus respectively, and distal reached the middle of inferior pubic ramus by corpora cavernosa angiography. In 14 health men by ultrasonic scanning, 6.60 cm in average from root of external penis to location of deep artery penis entered corpora cavernosa. Forty patients had been operated with the above methods. In the cases, the length of the penis varied from 0.5-4.0 cm in the flaccid, 2.0-6.0 cm in erect state before operation to 5.0-8.5 cm in the flaccid, 7.0-12.5 cm in erect state after operation. Twenty-eight cases had been followed up for more than one year, and 23 patients married with acceptable sexual life, eighteen men had successfully produced their next generations. The regenerative neurofibril could be seen under light microscope, immunohistochemistry marker, scanning and transmission electron microscope. The penis presented normal pain and touch feeling in the post-operating cases. The results showed that there was similar erectile ability of a normal male in the follow-up patients. CONCLUSION: With these basic and clinical researches, the restoration of penis tends to have a better appearance and function. It's a more optimal method compared with conventional procedures. The results have also provided valuable data for further study in related fields.

Enhancement of the primary flavor compound methional in potato by increasing the level of soluble methionine.

The primary flavor compound in potato, methional, is synthesized from methionine by the Strecker degradation reaction. A major problem associated with potato processing is the loss of methional. Methional or its precursor, methionine, is not added back during potato processing due to high costs of production. A novel approach to enhance the methional level in processed potato would be to increase the production of its precursor, soluble methionine (Met). Cystathionine gamma-synthase (CGS) is a key enzyme regulating methionine biosynthesis in plants. To increase the level of soluble methionine in potato, Arabidopsis thaliana CGS cDNA was introduced under transcriptional control of the cauliflower mosaic virus 35S promoter into Russet Burbank potato by Agrobacterium-mediated transformation. Ten different transgenic potato lines (CGS1-10) were analyzed. Immunoblot analysis demonstrated that Arabidopsis CGS is expressed in the leaves, tubers, and roots of transgenic potato plants. CGS enzymatic activity was higher in the leaves and roots of the transgenic potato lines compared to the wild-type potato. Methionine levels in the leaves, roots and tubers of transgenic potato lines were enhanced as high as 6-fold compared to those in wild type potato plants. The methional level in baked tubers of field-grown transgenic potato lines was increased between 2.4- and 4.4-fold in lines CGS1, CGS2, and CGS4. The increase observed in methional levels correlated with the soluble methionine level in the tubers from the same lines measured before processing. These results provide the first evidence that the methional level can be enhanced in processed potatoes by increasing the production of its precursor, methionine.