Peptide mixed phosphonates for covalent complex formation with thioesterases in nonribosomal peptide synthetases.

Natural macrocyclic peptides derived from microorganisms are medicinal resources that are important for the development of new therapeutic agents. Most of these molecules are biosynthesized by a nonribosomal peptide synthetase (NRPS). The thioesterase (TE) domain in NRPS is responsible for the macrocyclization of mature linear peptide thioesters in a final biosynthetic step. NRPS-TEs can cyclize synthetic linear peptide analogs and can be utilized as biocatalysts for the preparation of natural product derivatives. Although the structures and enzymatic activities of TEs have been investigated, the substrate recognition and substrate-TE interaction during the macrocyclization step are still unknown. To understand the TE-mediated macrocyclization, here we report the development of a substrate-based analog with mixed phosphonate warheads, which can react irreversibly with the Ser residue at the active site of TE. We have demonstrated that the tyrocidine A linear peptide (TLP) with a p-nitrophenyl phosphonate (PNP) enables efficient complex formation with tyrocidine synthetase C (TycC)-TE containing tyrocidine synthetase.

High-grade vaginal intraepithelial neoplasia after hysterectomy for high-grade cervical intraepithelial neoplasia: Is hysterectomy a "definitive" treatment compared to conization?

AIM: We investigated the frequency of early recurrence of vaginal intraepithelial neoplasia grade 2/3 (VaIN 2/3) (within 2 years) after hysterectomy for cervical intraepithelial neoplasia grade 3 (CIN3). The characteristics of the clinicopathological factors common to them were explored including different surgical methods. METHODS: As a retrospective observational study, a total of 647 CIN3 patients were divided into a conization and hysterectomy group (C group, n = 492; H group, n = 155), and HSIL (CIN2/3 or VaIN2/3) recurrence within 2 years after surgery was evaluated. A stratified analyses was performed. Surgical methods were divided into trans-abdominal, trans-vaginal, and laparoscopic. RESULTS: The recurrence of VaIN3 was detected in four cases (2.6%) in the H group, which was similar to that of CIN2/3 in the C group, 12 out of 491 patients (2.4%). The patients who developed VaIN3 were significantly older than those who did not (median, VaIN3: 71.0; VaIN1 and less: 48.0; p < 0.0001). All VaIN3 cases were detected within 5 months, although majority of cases were negative in the margin (3/4 cases; margin negative). The method of hysterectomy was not related to the VaIN3 recurrence. CONCLUSION: For CIN3 patients for whom hysterectomy is the main treatment, VaIN3 can develop in 2.6% within very shortly after operation even if surgical margin was negative. The elder the age, the higher the risk of early recurrence could be. Laparoscopic surgery is considered to be acceptable methods of hysterectomy.

Amine-catalyzed Synthesis of Fluorine-containing Polymers through Halogen Bonding.

Fluorine-containing polymers are one of the most useful materials among various polymers. In this study, we have developed synthesis methods for fluorine-containing polymers through sequential and chain polymerization based on the generation of perfluoroalkyl radicals by photoirradiation halogen bonding of perfluoroalkyl iodide and amines. In sequential polymerization, fluoroalkyl-alkyl-alternating polymers were synthesized by the polyaddition of diene and diiodoperfluoroalkane. In chain polymerization, polymers with perfluoroalkyl terminals were synthesized by polymerization of general-purpose monomers, with perfluoroalkyl iodide as the initiating species. Block polymers were also synthesized by successive chain polymerization to the polyaddition product.

Silkworm FoxL21 plays important roles as a regulator of ovarian development in both oogenesis and ovariole development.

The ovary is an important organ in reproduction. In insects, especially lepidopteran insects, the oocytes and reproductive organs develop rapidly during the pupal stage. Despite their drastic morphological changes, the molecular mechanisms of ovary development are not fully understood. In this study, it is found that forkhead box transcription factor L2, member 1 (FoxL21), which is known to be involved in ovarian differentiation and maintenance in vertebrates, is required for the development of the ovary in the silkworm, Bombyx mori. FoxL21 was expressed in the ovary and ovariole during the larval and pupal stage, respectively. In silkworms in which FoxL21 was knocked out by genome editing, multiple ovarian dysfunctions, such as, abnormal egg formation, thinning of the ovariole sheaths, and defective connection of the oviductus geminus with the ovariole were observed. Finally, ovarian transplantation experiments using the knockout silkworms revealed that FoxL21 functions in the ovariole, but not in the oviductus geminus.

A 36 kg Giant Ovarian Fibroma with Meigs Syndrome: A Case Report and Literature Review of Extremely Giant Ovarian Tumor.

Ovarian tumors can get extremely giant to occupy the whole abdominal cavity. We report a case of 36 kg solid ovarian tumor, which was the largest ovarian solid tumor that have been ever reported. A 54-year-old woman presented to our hospital with a chief complaint of markedly distended abdominal wall. Preoperative imaging examinations revealed that most of the tumor was uniform and its density was like that of subcutaneous fat. Pleural effusion was detected in the right thoracic region. We organized a multidisciplinary team and successfully resected the right adnexa. The patient had an uneventful postoperative course, and she was discharged on the 7th postoperative day and diagnosed with a fibroma of the ovary with Meigs syndrome. A comprehensive literature search revealed 48 cases of extremely giant ovarian tumor in these 20 years. Six out of 48 cases are solid. Twelve out of 48 cases are malignant or borderline malignant, and patients' age and tumor size/weight were not related to the frequency of malignancy/borderline malignancy. As many as 4 out of 48 patients died before their first hospital visit or early after surgery. Clinicians should consider a considerable high mortality and frequency of severe surgical complications when planning the treatment strategy for extremely giant ovarian tumors.

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

A defect in purine nucleotide metabolism in the silkworm, Bombyx mori, causes a translucent larval integument and male infertility.

p-oily (op) is a novel mutant of Bombyx mori exhibiting translucent larval integument and male infertility. Elucidation of the causative gene of the op mutant will help understand the genetic mechanism underlying larval integument coloration and male fertility. Using polymorphisms between B. mori and B. mandarina, the op locus was narrowed down to a 375-kb region. Using RNA-seq analysis, we found that op mutants have a frameshift mutation in the KWMTBOMO13770 gene located in the 375-kb region. A database search indicated that this gene is the human cytosolic 5'-nucleotidase II gene (cN-II) homolog in Bombyx, which mediates the conversion of inosine monophosphate (IMP) to inosine, a precursor of uric acid. CRISPR/Cas9-mediated knockout mutants of the Bm-cN-II gene showed translucent integuments, and there appeared translucent larvae in the crosses between knockout moths and +/op moths. Moreover, the translucent phenotype of, and decreased uric acid content in the larval integument caused by the mutations in the Bm-cN-II gene were rescued by oral administration of inosine. These results indicated that the Bm-cN-II gene is responsible for the op phenotype and that the molecular function of the Bm-cN-II gene is the conversion of IMP to inosine. We also discuss the genetic relationship between the Bm-cN-II gene and male fertility.

Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system.

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Induced by Repeated Forced Swimming in Mice.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by disabling fatigue of at least 6 months, in addition to symptoms such as muscle pain and muscle weakness. There is no treatment provides long-term benefits to most patients. Recently, clinical research suggested the involvement of pyruvate dehydrogenase (PDH) in ME/CFS. PDH is a crucial enzyme in the mitochondria matrix that links glycolysis to the tricarboxylic acid cycle and oxidative phosphorylation. However, it is little known whether PDH could be a therapeutic target. The purpose of this study was to establish ME/CFS in mice and to investigate the involvement of PDH in ME/CFS. To induce the chronic fatigue in mice, a repeated forced swimming test was conducted. To evaluate fatigue, we measured immobility time in forced swimming test and starting time of grooming. An open field test was conducted on day 8. After 25 d of the forced swimming test, the mitochondrial fraction in gastrocnemius muscle was isolated and PDH activity was measured. Moreover, we evaluated the effect of PDH activation by administering sodium dichloroacetate (DCA). In ME/CFS mice group, the immobility time and starting time of grooming increased time-dependently. In addition, the moved distance was decreased in ME/CFS mice. PDH activity was decreased in the mitochondrial fraction of the gastrocnemius muscle of the forced swimming group. DCA treatment may be beneficial in preventing fatigue-like behavior in ME/CFS. These findings indicate that ME/CFS model was established in mice and that a decrease in mitochondrial PDH activity is involved with the symptom of ME/CFS.

Sensorimotor gating deficits and effects of antipsychotics on the hyperactivity in VGF-overexpressing mice.

BACKGROUND: VGF nerve growth factor inducible (VGF) is a neuropeptide which is expressed in neuronal cells and endocrine cells. VGF is induced by several neurotrophic factors. The expression level of VGF in patients with schizophrenia is increased in cerebrospinal fluid (CSF) and prefrontal cortex. In our previous study, we generated mice in which the expression level of VGF in the brain was increased. VGF-overexpressing mice exhibited abnormal behaviors including hyperactivity. However, it remains unknown whether VGF-overexpressing mice exhibit the endophenotype of schizophrenia and whether abnormal behaviors in these mice can be improved by antipsychotics. METHODS: In the present study, we investigated schizophrenia-like behaviors and the responsiveness to antipsychotics in transgenic mice. RESULTS: VGF-overexpressing mice (1) exhibited prepulse inhibition (PPI) impairment, (2) showed normalized hyperactivity following antipsychotic drug treatment, and (3) showed abnormal responsiveness to haloperidol. CONCLUSION: Upregulation of VGF may be implicated in the pathophysiology of schizophrenia and abnormalities of dopaminergic signaling.

Substrate-dependent effects of molecular-targeted anticancer agents on activity of organic anion transporting polypeptide 1B1.

1. Organic anion-transporting polypeptide 1B1 (OATP1B1) plays an important role in the hepatic uptake of a broad range of substrate drugs. In vitro experiments show that molecular-targeted agents do not always have similar effects on OATP1B1 activity. 2. The purpose of this study was to clarify whether the effects of molecular-targeted agents on OATP1B1 are substrate-dependent. We used OATP1B1-transfected cells to compare the effects of molecular-targeted agents on OATP1B1-mediated uptake of fluorescein (FL), 2',7'-dichlorofluorescein (DCF), atorvastatin, SN-38 and valsartan. 3. Cabozantinib, cediranib, neratinib, pazopanib, regorafenib, sorafenib and tivantinib did not affect or only slightly affected OATP1B1-mediated substrate uptake. Nilotinib and lenvatinib moderately and strongly inhibited OATP1B1-mediated substrate uptake, respectively. In contrast, afatinib stimulated OATP1B1-mediated uptake of FL and SN-38, ceritinib stimulated that of valsartan, and nintedanib stimulated that of FL and valsartan. In addition, the effects of afatinib, ceritinib and nintedanib on OATP1B1 activity differed markedly depending on the type of substrate. Afatinib, ceritinib and nintedanib had a substrate-dependent effect on OATP1B1 activity. 4. We conclude that the evaluation of OATP1B1 activity using only a single probe substrate for some molecular-targeted agents may lead to a faulty understanding of their mechanisms of drug interactions.

Fermented Rice Germ Extract Alleviates Morphological and Functional Damage to Murine Gastrocnemius Muscle by Inactivation of AMP-Activated Protein Kinase.

Sarcopenia, loss of muscle mass and function, is mainly observed in elderly people. In this study, we investigated whether fermented rice germ extract (FRGE) has some effects on the mouse gastrocnemius muscle by using behavioral and morphological analyses, Western blotting, and a murine model of immobilization-induced muscle atrophy. Daily oral FRGE administration increased muscle weight and strength. In addition, myofiber size in gastrocnemius muscle of FRGE-treated mice was increased as revealed by morphological quantification. Activation of AMP-activated protein kinase (AMPK) signaling, which inhibits protein synthesis and stimulates protein degradation in gastrocnemius muscle, was significantly attenuated in the FRGE-treated mice compared with control mice. Expression level of forkhead box 3a (FOXO3a) protein was also significantly decreased in the FRGE-treated group. Moreover, the decrease in mean myofiber cross-sectional area in immobilized hindlimb in vehicle-treated mice was inhibited by FRGE treatment in histological analysis. In conclusion, FRGE increased the strength and weight of gastrocnemius muscle and myofiber size, and reduced immobilization-induced muscle atrophy in mice. These findings indicated that FRGE might be beneficial in preventing motor dysfunction in a range of conditions, including sarcopenia.